Materials and methods for the efficient production of acetate and other products

ABSTRACT

The subject invention provides materials and methods wherein unique and advantageous combinations of gene mutations are used to direct carbon flow from sugars to a single product. The techniques of the subject invention can be used to obtain products from native pathways as well as from recombinant pathways. In preferred embodiments, the subject invention provides new materials and methods for the efficient production of acetate and pyruvic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/703,812, filed Nov. 6, 2003, which claims the benefit of U.S.Provisional Application Ser. No. 60/424,372, filed Nov. 6, 2002.

The subject invention was made with government support under researchprojects supported by USDA/NRI, Grant No. 2001-35504-10669; USDA/IFAS,Grant No. 00-52104-9704; and USDOE Grant No. FG02-96ER20222. Thegovernment has certain rights in this invention.

BACKGROUND OF INVENTION

Recent trends toward the production of “green” chemicals will requiredevelopment of innovative synthesis techniques that are highly efficientand cost effective.

Throughout the past decade, a number of traditional chemical companiesin the United States and Europe have begun to develop infrastructuresfor the production of compounds using biocatalytic processes.Considerable progress has been reported toward new processes forcommodity chemicals such as ethanol (Ingram, L. O., H. C. Aldrich, A. C.C. Borges, T. B. Causey, A. Martinez, F. Morales, A. Saleh, S. A.Underwood, L. P. Yomano, S. W. York, J. Zaldivar, and S. Zhou, 1999“Enteric bacterial catalyst for fuel ethanol production” Biotechnol.Prog. 15:855-866; Underwood, S. A., S. Zhou, T. B. Causey, L. P. Yomano,K. T. Shanmugam, and L. O. Ingram, 2002 “Genetic changes to optimizecarbon partitioning between ethanol and biosynthesis in ethanologenicEscherichia coli.” Appl. Environ. Microbiol. 68:6263-6272), lactic acid(Zhou, S., T. B. Causey, A. Hasona, K. T. Shanmugam and L. O. Ingram,2003 “Production of optically pure D-lactic acid in mineral salts mediumby metabolically engineered Escherichia coli W3110” Appl. Environ.Microbiol. 69:399-407; Chang, D., S. Shin, J. Rhee, and J. Pan, 1999“Homofermentative production of D- or L-lactate in metabolicallyengineered Escherichia coli RR1” Appl. Environ. Microbiol. 65:1384-1389;Dien, B. S., N. N. Nichols, and R. J. Bothast, 2001 “RecombinantEscherichia coil engineered for the production of L-lactic acid fromhexose and pentose sugars” J. Ind. Microbiol. Biotechnol. 27:259-264),1,3-propanediol (Nakamura, U.S. Pat. No. 6,013,494; Tong, I., H. H.Liao, and D.C. Cameron, 1991 “1,3-propanediol production by Escherichiacoli expressing genes from the Klebsiella-pneumoniae-DHA regulon” App.Env. Microbiol. 57:3541-3546), and adipic acid (Niu, W., K. M. Draths,and J. W. Frost, 2002 “Benzene-free synthesis of adipic acid”Biotechnol. Prog 18:201-211).

In addition, advances have been made in the genetic engineering ofmicrobes for higher value specialty compounds such as acetate,polyketides (Beck, B. J., C. C. Aldrich, R. A. Fecik, K. A. Reynolds,and D. H. Sherman, 2003 “Iterative chain elongation by a pikromycinmonomodular polyketide synthase” J. Am. Chem. Soc. 125:4682-4683; Dayem,L. C., J. R. Carney, D. V. Santi, B. A. Pfeifer, C. Khosla, and J. T.Kealey, 2002 “Metabolic engineering of a methylmalonyl-CoAmutase—epimerase pathway for complex polyketide biosynthesis inEscherichia coli.” Biochem. 41:5193-5201) and carotenoids (Wang,Chia-wei, Min-Kyu Oh, J. C. Liao, 2000 “Directed evolution ofmetabolically engineered Escherichia coli for carotenoid production”Biotechnol. Prog. 16:922-926).

Acetic acid, a widely used specialty chemical in the food industry, hasrecently emerged as a potential bulk chemical for the production ofplastics and solvents. Acetic acid has been produced using microbialsystems; however, the production of acetic acid in microbial systemscompetes with the production of CO₂ and cell mass. Thus, while efficientacetate-producing microbial systems are important for industrial uses,the systems must have an increased output of acetate with a decreasedinput of expensive microbial nutrients.

The biological production of acetic acid has been largely displaced bypetrochemical routes as the uses for this commodity chemical haveexpanded from food products to plastics, solvents, and road de-icers(Freer, S. N., 2002 “Acetic acid production by Dekkera/Brettanomycesyeasts” World J. Microbiol. Biotechnol. 18:271-275). In 2001, the worldproduction of acetic acid reached an estimated 6.8 million metric tons,half of which was produced in the United States.

Previously, three microbial approaches have been explored for aceticacid production. In the two-step commercial process, sugars arefermented to ethanol by Saccharomyces yeast. Then, the resulting beersare oxidized to acetic acid by Acetobacter under aerobic conditions(Berraud, C., 2000 “Production of highly concentrated vinegar infed-batch culture” Biotechnol. Lett. 22:451-454; Cheryan, M., S. Parekh,M. Shah, and K. Witjitra, 1997 “Production of acetic acid by Clostridiumthermoaceticum” Adv. Appl. Microbiol. 43:1-33). Using this process,acetic acid titres of around 650 mM are typically produced; however,higher titres can be readily achieved by the addition of complexnutrients in fed-batch processes requiring 60-120 hours. Overall yieldsfor this commercial process have been estimated to be 76% of thetheoretical maximum (2 acetate per glucose; 0.67 g acetic acid per gglucose).

Under a second approach, carbohydrates can be anaerobically metabolizedto acetic acid at substantially higher yields (3 acetates per glucose)by Clostridia that contain the Ljungdahl-Wood pathway for acetogenesis(Berraud, C., 2000 “Production of highly concentrated vinegar infed-batch culture” Biotechnol. Lett. 22:451-454; Ljungdahl, L. G., 1986“The autotrophic pathway of acetate synthesis in acetogenic bacteria”Ann. Rev. Microbiol. 40:415-450). In particular, Clostridiumthermoaceticum containing the Ljungdahl-Wood pathway produces highyields of acetic acid (Cheryan, M., S. Parekh, M. Shah, and K. Witjitra,1997 “Production of acetic acid by Clostridium thermoaceticum” Adv.Appl. Microbiol. 43:1-33).

Recently, Freer (Freer, S. N., 2002 “Acetic acid production byDekkera/Brettanomyces yeasts” World J. Microbiol. Biotechnol.18:271-275) identified yeast strains (Dekkera and Brettanomyces) thatproduce acetic acid as a primary product from glucose for potential usein acetic acid production. All three of these current microbial aceticacid production systems require complex nutrients, which increase thecost of materials, acetate purification, and waste disposal.

Escherichia coli is widely used as a biocatalyst for high value productssuch as recombinant proteins (Akesson, M., P. Hagander, and J. P.Axelsson, 2001 “Avoiding acetate accumulation in Escherichia colicultures using feedback control of glucose feeding” Biotechnol. Bioeng.73:223-230; Aristidou, A. A., K. San, and G. N. Bennett, 1995 “Metabolicengineering of Escherichia coli to enhance recombinant proteinproduction through acetate reduction” Biotechnol. Prog. 11:475-478;Contiero, J., C. Beatty, S. Kumar, C. L. DeSanti, W. R. Strohl, and A.Wolfe, 2000 “Effects of mutations in acetate metabolism onhigh-cell-density growth of Escherichia coli” J. Ind. Microbiol.24:421-430; Luli, G. W. and R. Strohl, 1990 “Comparison of growth,acetate production and acetate inhibition of Escherichia coli strains inbatch and fed-batch fermentations” Appl. Environ. Microbiol.56:1004-1011) and amino acids (Chotani, G., T. Dodge, A. Hsu, M. Kumar,R. LaDuca, D. Trimbur, W. Weyler, and K. Sanford, 2000 “The commercialproduction of chemicals using pathway engineering” Biochem. Biophys.Acta 1543:434-455; Eggeling, L., W. Pfefferle, and H. Sahm, 2001 “Aminoacids,” p. 281-304 in C. Ratledge and B. Kristiansen (ed.), BasicBiotechnology, 2^(nd) edition. Cambridge University Press. Cambridge,U.K.).

Escherichia coli generate acetyl˜CoA during fermentative and oxidativemetabolism, which the cell then uses to produce small amounts of acetate(Akesson, M., P. Hagander, and J. P. Axelsson, 2001 “Avoiding acetateaccumulation in Escherichia coli cultures using feedback control ofglucose feeding” Biotechnol. Bioeng. 73:223-230; Contiero, J., C.Beatty, S. Kumar, C. L. DeSanti, W. R. Strohl, and A. Wolfe, 2000“Effects of mutations in acetate metabolism on high-cell-density growthof Escherichia coli”, J. Ind. Microbiol. 24:421-430).

Many E. coli strains grow well in simple mineral salts medium andreadily metabolize all of the hexose and pentose sugar constituents ofplant biomass (Ingram, L. O., H. C. Aldrich, A. C. C. Borges, T. B.Causey, A. Martinez, F. Morales, A. Saleh, S. A. Underwood, L. P.Yomano, S. W. York, J. Zaldivar, and S. Zhou, 1999 “Enteric bacterialcatalyst for fuel ethanol production” Biotechnol. Prog. 15:855-866).During aerobic and anaerobic carbohydrate metabolism, acetate istypically produced as a minor product. Recent successes have beenreported in the engineering of E. coli strains for commodity chemicalssuch as propanediol (Nakamura, C. E., A. A. Gatenby, Hsu, A. K.-H., R.D. LaReau, S. L. Haynie, M. Diaz-Torres, D. E. Trimbur, G. M. Whited, V.Nagarajan, M. S. Payne, S. K. Picataggio, and R. V. Nair, 2000 “Methodfor the production of 1,3-propanediol by recombinant microorganisms”U.S. Pat. No. 6,013,494; Tong, I., H. H. Liao, and D. C. Cameron, 1991“1,3-propanediol production by Escherichia coli expressing genes fromthe Klebsiella-pneumoniae-DHA regulon” App. Env. Microbiol.57:3541-3546), adipic acid (Niu, W., K. M. Draths, and J. W. Frost, 2002“Benzene-free synthesis of adipic acid” Biotechnol. Prog. 18:201-211),lactic acid (Chang, D., S. Shin, J. Rhee, and J. Pan, 1999“Homofermentative production of D- or L-lactate in metabolicallyengineered Escherichia coli RR1” Appl. Environ. Microbiol. 65:1384-1389;Dien, B. S., N. N. Nichols, and R. J. Bothast, 2001 “RecombinantEscherichia coli engineered for the production of L-lactic acid fromhexose and pentose sugars” J. Ind. Microbiol. Biotechnol. 27:259-264),succinic acid (Donnelly, M. I., C. Sanville-Millard, and R. Chatterjee,1998 “Method for construction of bacterial strains with increasedsuccinic acid production” U.S. Pat. No. 6,159,738; Vemuri, G. N., M. A.Altman, and E. Altman, 2002 “Effects of growth mode and pyruvatecarboxylase on succinic acid production by metabolically engineeredstrains of Escherichia coli” J. Bacteriol. 68:1715-1727), and ethanol(Ingram, L. O., H. C. Aldrich, A. C. C. Borges, T. B. Causey, A.Martinez, F. Morales, A. Saleh, S. A. Underwood, L. P. Yomano, S. W.York, J. Zaldivar, and S. Zhou, 1999 “Enteric bacterial catalyst forfuel ethanol production” Biotechnol. Prog. 15:855-866). In using theseaerobic and anaerobic processes, the resultant production of acetate bythe native pathway (phosphotransacetylase and acetate kinase) hasgenerally been regarded as an undesirable consequence of excessiveglycolytic flux (Akesson, M., P. Hagander, and J. P. Axelsson, 2001“Avoiding acetate accumulation in Escherichia coli cultures usingfeedback control of glucose feeding” Biotechnol. Bioeng. 73:223-230;Aristidou, A. A., K. San, and G. N. Bennett, 1995 “Metabolic engineeringof Escherichia coli to enhance recombinant protein production throughacetate reduction” Biotechnol. Prog. 11:475-478; Contiero, J., C.Beatty, S. Kumar, C. L. DeSanti, W. R. Strohl, and A. Wolfe, 2000“Effects of mutations in acetate metabolism on high-cell-density growthof Escherichia coli” J. Ind. Microbiol. 24:421-430; Farmer, W. R., andJ. C. Liao, 1997 “Reduction of aerobic acetate production by Escherichiacoli 1997” Appl. Environ. Microbiol. 63:3205-3210).

Chao and Liao (Chao, Y., and J. C. Liao, 1994 “Metabolic responses tosubstrate futile cycling in Escherichia coli” J. Biol. Chem.269:5122-5126) and Patnaik et al. (Patnaik, R., W. D. Roof, R. F. Young,and J. C. Liao, 1992 “Stimulation of glucose catabolism in Escherichiacoli by a potential futile cycle” J. Bacteriol. 174:7525-7532)demonstrated a 2-fold stimulation of glycolytic flux in E. coli usingplasmids to express genes that created futile cycles to consume ATP.

Recently, Koebmann et al. (Koebmann, B. J., H. V. Westerhoff, J. L.Snoep, D. Nilsson, and P. R. Jensen, 2002 “The glycolytic flux inEscherichia coli is controlled by the demand for ATP” J. Bacteriol.184:3909-3916) independently concluded that glycolytic flux is limitedby ATP utilization during the oxidative metabolism of glucose. In theirstudies, flux increased in a dose-dependent manner with controlledexpression of F₁ ATPase from a plasmid. Thus glycolytic flux appears tobe regulated by the economy of supply and demand as proposed by Hofmeyrand Cornish-Bowden (Hofmeyer, J.-H. S., and A. Cornish-Bowden, 2000“Regulating the cellular economy of supply and demand” FEBS Lett.467:47-51).

Currently, only the two-part commercial process, the Ljungdahl-Woodpathway-containing Clostridia, as well as special yeast strains havebeen investigated as potential biocatalysts for the production ofacetate. Due to the competing production of dicarboxylic acids and cellmass from glucose, the level of acetate production using these methodshas been relatively low. Indeed, none of these methods have beenreported to grow and produce acetate efficiently in mineral salts mediacontaining sugar. In fact, each of these methods requires the use ofcomplex nutrients, which ultimately increases the cost of materials,acetate purification, and waste disposal. Therefore, a need remains forbetter biocatalysts that efficiently produce acetate and otherfermentation products using a mineral salts medium.

Pyruvic acid is currently manufactured for use as a food additive,nutriceutical, and weight control supplement (Li, Y., J. Chen, and S.-Y.Lun, 2001 “Biotechnological production of pyruvic acid” Appl. Microbiol.Biotechnol. 57:451-459). Pyruvic acid can also be used as a startingmaterial for the synthesis of amino acids such as alanine, tyrosine,phenylalanine, and tryptophan and for acetaldehyde production.

Pyruvate is produced commercially by both chemical and microbialprocesses. Chemical synthesis involves the decarboxylation anddehydration of calcium tartrate, a by-product of the wine industry. Thisprocess involves toxic solvents and is energy intensive with anestimated production cost of $8,650 per ton of pyruvate. Microbialpyruvate production is based primarily on two microorganisms, amulti-vitamin auxotroph of the yeast Torulopsis glabrata (Li, Y., J.Chen, and S.-Y. Lun, and X. S. Rui, 2001 “Efficient pyruvate productionby a multi-vitamin auxotroph of Torulopsis glabrata: key role andoptimization of vitamin levels” Appl. Microbiol. Biotechnol. 55:680-68)and a lipoic acid auxotroph of Escherichia coli containing a mutation inthe F₁ ATPase component of (F₁F₀)H⁺-ATP synthase (Yokota, A., Y.Terasawa, N. Takaoka, H. Shimizu, and F. Tomita, 1994 “Pyruvic acidproduction by an F₁-ATPase-defective mutant of Escherichia coliW1485lip2” Biosci. Biotech. Biochem. 58:2164-2167). Both of theseproduction strains require precise regulation of media compositionduring fermentation and complex supplements. The estimated productioncosts of pyruvate production by microbial fermentation with thesestrains is estimated to be 14.5% ($1,255 per ton pyruvate) of that forchemical synthesis.

Recently, Tomar et al. (Tomar, A., M. A. Eiteman, and E. Altman, 2003“The effect of acetate pathway mutations on the production of pyruvatein Escherichia coli.” Appl. Microbiol. Biotechnol. 62:76-82.2003) havedescribed a new mutant strain of E. coli for pyruvate production. Thisstrain contains three mutations, ppc (phosphoenolpyruvate carboxylase),aceF (pyruvate dehydrogenase), and adhE (alcohol dehydrogenase) and iscapable of producing 0.65 grams pyruvate per gram of glucose usingcomplex media supplemented with acetate.

Typical production rates of pyruvate for biocatalysts are around 1 g L⁻¹h⁻¹ with yields exceeding half the weight of substrate. Torulopsisglabrata, the yeast strain currently used for the commercial productionof pyruvate, can achieve pyruvate titers of 69 g L⁻¹. As noted above, T.glabrata strains used in the commercial process are multivitaminauxotrophs requiring tight regulation of vitamin concentrations whichresult in complex vitamin feeding strategies during fermentation (Li,Y., J. Chen, and S.-Y. Lun, 2001 “Biotechnological production of pyruvicacid” Appl. Microbiol. Biotechnol. 57:451-459). Previous E. coli strainsconstructed for pyruvate production were cultured in complex media andhave been plagued by low titers and yields (Tomar, A. et al. 2003, “Theeffect of acetate pathway mutations on the production of pyruvate inEscherichia coli.” Appl. Microbiol. Biotechnol. 62:76-82; Yokota A. etal., 1994 “Pyruvic acid production by an F₁-ATPase-defective mutant ofEscherichia coli W1485lip2.” Biosci. Biotech. Biochem. 58:2164-6167).

Nutrients in culture medium often represent a major cost associated withcommercial fermentations. The use of a mineral salts medium andinexpensive carbon source offers the potential to improve the economicsof many biological processes by reducing the costs of materials, productpurification, and waste disposal (Zhang, J. and R. Greasham, 1999. Appl.Microbiol. Biotechnol. 51:407-421).

There is a need in the art to identify and develop new, efficient, andenvironmentally friendly processes for producing specialty compounds.

BRIEF SUMMARY

The subject invention provides materials and methods wherein unique andadvantageous combinations of gene mutations are used to direct carbonflow from sugars to a desired product. The techniques of the subjectinvention can be used to obtain products from native pathways as well asfrom recombinant pathways.

The materials and methods of the subject invention can be used toproduce a variety of products with only mineral salts and sugar asnutrients. Useful products include pure acetic acid; 1,3-propanediol;2,3-propanediol; pyruvate; dicarboxylic acids; adipic acid; amino acids,including aliphatic and aromatic amino acids; and alcohols includingethanol, butanol, isopropanol, and propanol. In preferred embodiments,the subject invention provides new materials and methods for theefficient production of acetate. In further preferred embodiments, thesubject invention provides advantageous biocatalysts for acetateproduction and for pyruvate production.

In a specific embodiment, the subject invention provides a recombinantderivative of Escherichia coli W3110 that contains six chromosomaldeletions (focA-pflB frdBC ldhA atpFH sucA adhE). The resulting strain(TC36) exhibits approximately a 2-fold increase in maximal rates ofacetate production (specific and volumetric) over W3110. This increasecan be attributed to the mutation in the (F₁F₀)H⁺-ATP synthase, whicheliminates ATP production by oxidative phosphorylation while retainingcytoplasmic F₁-ATP synthase for the gratuitous consumption of ATP.

TC36 produces acetic acid in mineral salts medium containing glucosewith a yield of 68% of the maximum theoretical yield using nativepathways (two acetates per glucose). Advantageously, TC36 is devoid ofplasmids and antibiotic resistance genes.

Further embodiments of the subject invention provide additionalderivatives of Escherichia coli W3110 as new biocatalysts for theproduction of homo-acetate. In one embodiment, homo-acetate productionby the new strain, TC36, approaches the theoretical maximum of twoacetates per glucose. Eliminating the fermentation pathways of W3110resulted in the new strain SZ47 and doubled the loss of carbon asvolatile products. While the rate of acetate production decreased inSZ47 as compared to W3110, the cell yield increased. The inactivation ofoxidative phosphorylation (ΔatpFH) in SZ47 to produce TC24 resulted in a5-fold increase in acetate yield and a 3-fold improvement in carbonrecovery.

In accordance with the subject invention, competing pathways areeliminated by chromosomal inactivation of genes encoding lactatedehydrogenase, pyruvate formatelyase, and fumarate reductase(Δ(focA-pflB)::FRT ΔfrdBC ΔldhA), (F₁F₀)H⁺-ATP synthase (atpFH),alcohol/aldehyde dehydrogenase (adhE), and 2-ketoglutarate dehydrogenase(sucA), which increases the production of acetate. Using a simpletwo-step batch feeding strategy can increase acetate production.

Specifically, a second addition of 3% glucose added at the end of thegrowth phase (12 h) and metabolized to completion results in 78% of thetheoretical maximum. A further increase in acetate production can beobtained by combining the two-step batch feeding strategy with anitrogen limitation, which results in 86% of the theoretical maximum.

The subject invention provides a method to reduce the loss of substratecarbon into cell mass and/or into carbon dioxide. Also, the subjectinvention provides a method to reduce oxygen demand during bioconversionprocess.

The use of mineral salts medium, lack of antibiotic resistance genes orplasmids, high yield of homo-acetate, and high product purity achievedaccording to the subject invention are advantageous because of reducedcosts associated with nutrients, purification, containment, BOD, andwaste treatment.

In an alternative embodiment, the subject invention provides a newbiocatalyst for the efficient production of pyruvate from glucose thatrequires only simple mineral salts as nutrients.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Diagram summarizing genetic modifications used to redirectglucose metabolism to acetate. Bold arrows mark principle metabolicroutes in TC36. Reactions which have been blocked by gene deletions inTC36 are marked with solid circles. Genes encoding enzymes are shown initalics. A. Central carbon metabolism. Bold arrows indicate the primarypathway for acetate production from glucose in TC36. This strainproduces a net of 4 ATP equivalents (˜P) per glucose molecule. B.Oxidative phosphorylation. The ATPsynthase is inactive in TC36 althoughthe electron transport system remains functional as the primary routefor NADH oxidation in TC36 (bold arrows). C. F₁-ATPase remains active inTC36 for the regeneration of ADP but lacks subunits for membraneassembly.

FIG. 2 Diagram summarizing plasmid constructions.

FIG. 3 Effects of selected mutations on growth (A), glucose utilization(B), and base consumption (C). Symbols: ▪, W3110 (wild type); □,SZ47(Δ(focA-pflB)::FRT ΔfrdBC ΔldhA); ◯, TC24(Δ(focA-pflB)::FRT ΔfrdBCΔldhA Δatp(FH)::FRT); , TC36 (succ⁺; Δ(focA-pflB)::FRT ΔfrdBC ΔldhAΔatp(FH)::FRT ΔadhE::FRT ΔsucA::FRT).

FIG. 4 Effects of selected mutations on the production of acetate (A),dicarboxylic acids (B), and pyruvate (C). Symbols: ▪, W3110 (wild type);□, SZ47(Δ(focA-pflB)::FRT ΔfrdBC ΔldhA); ◯, TC24(Δ(focA-pflB)::FRTΔfrdBC ΔldhA Δatp(FH)::FRT); , TC36 (Succ⁺; Δ(focA-pflB)::FRT ΔfrdBCΔldhA Δatp(FH)::FRT ΔadhE::FRT ΔsucA::FRT).

FIG. 5 Fermentation of 6% glucose to acetate by TC36 in mineral saltsmedium. Fermentation was begun with 3% glucose followed by a secondaddition of 3% glucose after 12 h. Symbols: □, cell mass; ◯, glucose; ,acetate.

FIG. 6 Summary of central metabolism in E coli. A. Carbon metabolism. B.Oxidative phosphorylation. C. Cytoplasmic F₁ATPase subunit (active).

FIG. 7 Effect of oxygen level on pyruvate production by TC36. Cells wereinoculated into fermentation broth at 100% air saturation andcontinuously sparged with air until the oxygen levels declined to 5%saturation. At this time, oxygen was blended to maintain 5% saturationduring the remaining period of incubation (open symbols). Alternatively,media was sparged with a mixture of air and nitrogen to provide 5% airsaturation prior to inoculation and sparging switched to air and oxygenas needed to maintain 5% air saturation (closed symbols).

FIG. 8 Batch fermentation of glucose by mutant strains of E. coli. A.Cell growth; B. Glucose utilization; C. Acetate production; D. Pyruvateproduction.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a sense primer used according to the subject invention.

SEQ ID NO:2 is an antisense primer used according to the subjectinvention.

DETAILED DISCLOSURE

The subject invention provides materials and methods wherein unique andadvantageous combinations of gene mutations are used to direct carbonflow to a desired product. The techniques of the subject invention canbe used to obtain products from native pathways as well as fromrecombinant pathways.

Advantageously, the subject invention provides a versatile platform forthe production of a variety of products with only mineral salts andsugar as nutrients. Useful products include pure acetic acid;1,3-propanediol; 2,3-propanediol; pyruvate; dicarboxylic acids; adipicacid; and amino acids, including aliphatic and aromatic amino acids. Inpreferred embodiments, the subject invention provides new materials andmethods for the efficient production of acetate.

In preferred embodiments, the subject invention provides strains of E.coli (lacking plasmids and antibiotic resistance genes) as biocatalystsfor the production of chemically pure acetate and/or pyruvate. Unlikeother acetate-producing microbial systems, the subject invention canemploy a single step process using sugars as substrates, high rates ofacetate production (almost two-fold higher), high acetate yields, simplenutrition requirements (mineral salts medium), and a robust metabolismpermitting the bioconversion of hexoses, pentoses, and manydissacharides.

Specifically exemplified herein is a new E. coli biocatalyst containingsix chromosomal deletions (ΔfocApflB ΔfrdCD ΔldhA ΔatpFH ΔsucA ΔadhE).The resulting strain (TC36) contains no plasmids or antibioticresistance genes and produces high yields of acetate from glucose in amineral salts medium.

Further embodiments of the subject invention provide additionalderivatives of Escherichia coli W3110 as new biocatalysts for theproduction of acetate. Eliminating the fermentation pathways of W3110resulted in the new strain SZ47 and doubled the loss of carbon asvolatile products. While the rate of acetate production decreased inSZ47 as compared to W3110, the cell yield increased. The inactivation ofoxidative phosphorylation (ΔatpFH) in SZ47 to produce TC24 resulted in a5-fold increase in acetate yield and a 3-fold improvement in carbonrecovery. Homo-acetate production by the new strain, TC36, approachesthe theoretical maximum of two acetates per glucose.

The methods of the subject invention are particularly advantageousbecause, in a preferred embodiment, deletions (rather than mutationswhich simply change a sequence) are used to inactivate pathways.Deletions provide maximum stability; with deletions, there is noopportunity for a reverse mutation to restore function. Please note,however, that as used herein, “mutations” includes changes in sequenceor deletions unless the context clearly indicates otherwise. Suchchanges or deletions in polynucleotide sequences are also referred toherein as genetic “modifications.”

For optimal acetate production in accordance with a specific embodimentof the subject invention, deletions in W3110 that inactivate oxidativephosphorylation (ΔatpFH), disrupt the cyclic function of thetricarboxylic acid cycle (ΔsucA), and eliminate all major fermentationpathways (ΔfocA-pflB, ΔfrdBC, ΔldhA, ΔadhE) are combined. One suchstrain, TC36, metabolizes sugars to acetate with the efficiency offermentative metabolism, diverting a minimum of carbon to cell mass(biocatalyst) and CO₂, which results in extremely high product yields.

For improved acetic acid yields, a simple two-step batch feedingstrategy can be used in which a second addition of 3% glucose is addedat the end of the growth phase (12 h). Further improved acetic acidyields can be obtained by combining this two-step batch feeding strategywith a nitrogen limitation.

Although production of homo-acetate using a recombinant gene isspecifically exemplified herein, those skilled in the art having thebenefit of the subject disclosure could utilize other genes (singlegenes or combinations), to produce alternative oxidized or reducedproducts.

The choice of genes for inactivation of competing fermentation pathways,as described herein, is important to maximize yield and minimizenutritional requirements. For example, carbohydrates can beanaerobically metabolized to acetic acid at substantially higher yields(3 acetates per glucose) by Clostridia (anaerobic) that contain theLjungdahl-Wood pathway for acetogenesis (Berraud, C., 2000 “Productionof highly concentrated vinegar in fed-batch culture” Biotechnol. Lett.22:451-454; Ljungdahl, L. G., 1986, “The autotrophic pathway of acetatesynthesis in acetogenic bacteria” Ann. Rev. Microbiol. 40:415-450).Specifically, Clostridium thermoaceticum containing the Lungdahl-Woodpathway produce higher yields of acetate than TC36 (Cheryan, M., S.Parekh, M. Shah and K. Witjitra, 1997 “Production of acetic acid byClostridium thermoaceticum” Adv. Appl. Microbiol. 43:1-33). As well,maximum titres with TC36 are lower than can be achieved by ethanoloxidation using Acetobacter in the two-step commercial process (Berraud,C., 2000 “Production of highly concentrated vinegar in fed-batchculture” Biotechnol. Lett. 22:451-454). However, the specific genedeletions of TC36 lead to acetate production rates almost two-foldhigher than either of the aforementioned processes and require onlymineral salts as nutrients.

E. coli TC36 offers a unique set of advantages over currently employedbiocatalysts for the commercial production of acetate: a single stepprocess using sugars as substrates, high rates of acetate production,high acetate yields, simple nutrition (mineral salts medium), and arobust metabolism permitting the bioconversion of hexoses, pentoses, andmany dissacharides.

In an alternative embodiment, the subject invention provides a newbiocatalyst for the efficient production of pyruvate from glucose thatrequires only simple mineral salts as nutrients.

As discussed herein, in a preferred embodiment, the materials andmethods of the subject invention provide at least the followingadvantages:

1. The ability to convert hexose and pentose sugars to acetate at veryhigh carbon efficiency in mineral salts medium without the addition ofcomplex nutrients.

2. The lack of plasmids, which may be lost during scale up. This resultsin a simplified process at less cost.

3. The absence of a need for antibiotic selection. This provides costand public health advantages.

4. The absence of antibiotic resistance genes. This also provides apublic health advantage.

Production of Acetate

Genetically modified E. coli W3110 was developed to produce acetic acidas the primary product from glucose during aerobic growth using onlymineral salts as nutrients. The resulting biocatalyst (TC36) containsmultiple chromosomal alterations (FIG. 1) that direct carbon flow toacetate and minimize carbon loss to cell mass, CO₂, and alternativeproducts. Strain TC36 is devoid of plasmids and antibiotic resistancegenes, both potential advantages for commercial use. The subjectinvention provides an additional derivative of Escherichia coli W3110 asa new biocatalyst for the production of homo-acetate. Acetate productionby this new strain (TC36) approaches the theoretical maximum of twoacetate per glucose due to the disruption of oxidative phosphorylation.

Chromosomal deletions were used instead of point mutations to maximizestability. All antibiotic resistance genes and auxotrophic requirementswere eliminated to permit growth in simple mineral salts medium. Duringoxidative metabolism, up to half of the substrate carbon can beconverted to roughly equal amounts of cell mass and CO₂ (Contiero, J.,C. Beatty, S. Kumar, C. L. DeSanti, W. R. Strohl, and A. Wolfe, 2000“Effects of mutations in acetate metabolism on high-cell-density growthof Escherichia coli” J. Ind. Microbiol. 24:421-430; Neidhardt, F. C., J.L. Ingraham, and M. Schaechter, 1990 “Physiology of the bacterial cell:A molecular approach” Sinauer Associates, Inc., Sunderland, Mass.;Varma, A., B. W. Boesch, and B. O. Palsson, 1993 “Stoichiometricinterpretation of Escherichia coli glucose catabolism under variousoxygenation rates” Appl. Environ. Microbiol. 59:2465-2473) with minimalcarbon flow into alternative products, such as acetate.

To reduce the opportunity for excessive growth during oxidativemetabolism, ATP production from NADH oxidation (oxidativephosphorylation) can be eliminated (or substantially reduced) bydeleting the portion of (F₁F₀)H⁺-ATP synthase involved in membraneassembly while preserving a functional cytoplasmic F₁-ATPase to providegratuitous hydrolysis of ATP. With this mutation, a maximum of 4 ATPmolecules (net) can be produced per glucose (assumes all pyruvate ismetabolized to acetyl˜CoA and acetate) as compared to a theoreticalmaximum of 33 ATP molecules for wild-type strains of E. coli.Substantial reduction refers to a greater than 80% reduction.

Excessive oxidation of substrate to CO₂ and NADH production wereeliminated by disrupting the cyclic function of the tricarboxylic acidcycle (ΔsucA) with the added benefit of reducing oxygen demand for NADHoxidation. Additional mutations were introduced to eliminate all majorfermentation pathways as alternative routes for NADH oxidation,minimizing the formation of alternative products. The resulting strain,TC36, has absolute requirements for substrate level phosphorylation andfor an external electron acceptor that can couple to the electrontransport system during growth in mineral salts medium to maintain redoxbalance. With genetic blocks in all major fermentation pathways andoxidative phosphorylation, this strain is relatively insensitive tovariations in dissolved oxygen.

The (F₁F₀)H⁺-ATP synthase and 2-ketoglutarate dehydrogenase mutationsintroduced into TC36 to minimize the levels of ATP and NAD(P)H fromglucose under oxidative conditions would also be expected to promoteglycolysis through native allosteric controls (Neidhardt, F. C., J. L.Ingraham, and M. Schaechter, 1990 “Physiology of the bacterial cell: Amolecular approach” Sinauer Associates, Inc., Sunderland, Mass.;Underwood, S. A., M. L. Buszko, K. T. Shanmugam, and L. O. Ingram, 2002“Flux through citrate synthase limits the growth of ethanologenicEscherichia coli KO11 during xylose fermentation” Appl. Environ.Microbiol. 68:1071-1081), providing a mechanism for the observed 2-foldincrease in glycolytic flux as compared to W3110 (wild type).

With additional mutations in fermentation pathways, further metabolismof pyruvate was limited primarily to small biosynthetic needs andconversion to acetyl˜CoA by the pyruvate dehydrogenase complex. Althoughpyruvate dehydrogenase is activated by low NADH, acetyl˜CoA productionmay be limited by the availability of free CoA (note pyruvateaccumulation in TC36 broth between 9 h and 15 h; FIG. 4C). Resultingrises in pyruvate pools would serve as an allosteric activator ofphosphotransferase (Suzuki, T., 1969 “Phosphotransacetylase ofEscherichia coli B, activation by pyruvate and inhibition by NADH andcertain nucleotides” Biochim. Biophys. Acta 191:559-569), the firstcommitted step for acetate production from acetyl˜CoA. Gratuitous ATPhydrolysis by F1-ATPase should ensure the availability of ADP for thefinal step in acetate production catalyzed by acetate kinase (FIG. 1).Excess pyruvate can also be directly oxidized to acetate by pyruvateoxidase (poxB), an enzyme that is induced during the latter stages ofgrowth and by environmental stress (Chang, Y.-Y., A.-Y. Wang, and J. E.Cronan, Jr., 1994 “Expression of Escherichia coli pyruvate oxidase(PoxB) depends on the sigma factor enocoded by the rpoS (katF) gene”Mol. Microbiol. 11:1019-1028). This enzyme may also contribute toacetate production by TC36.

Eliminating oxidative phosphorylation while preserving F₁ ATPaseresulted in a 2-fold increase in glycolytic flux (TC24 and TC36).

In a specific embodiment, the subject invention utilizes strategies thatdelete subunits concerned with the membrane assembly of the (F₁F₀)H⁺-ATPsynthase, create futile cycles for ATP consumption, or increasecytoplasmic levels of the ATPase activities, to decrease cell yield,increase metabolic flux, and increase product yield in bioconversionprocesses.

Strain TC36 can be used as a biocatalysis platform for the efficientproduction of oxidized products. Under conditions of glucose excess,strain TC36 produced a maximum of 878 mM acetate, 75% of the maximumtheoretical yield or 0.50 g acetate per g glucose. Only cell mass andsmall amounts of organic acids were produced as co-products withacetate. It is likely that 878 mM acetate approaches the upper limit oftolerance for the metabolism in TC36. Concentrations as low as 50 mMacetate have been shown to induce a stress response in E. coli(Kirkpatrick, C., L. M. Maurer, N. E. Oyelakin, Y. N. Yoncheva, R.Maurer, and J. L. Slonczewski, 2001 “Acetate and formate stress:Opposite responses in the proteomes of Escherichia coli” J. Bacteriol.183:6466-6477). The minimal inhibitory concentration for growth has beenpreviously reported as 300-400 mM acetate at neutral pH (Lasko, D. R.,N. Zamboni, and U. Sauer, 2000 “Bacterial response to acetate challenge:a comparison of tolerance among species” Appl. Microbiol. Biotechnol.54:243-247; Zaldivar, J., and L. O. Ingram, 1999 “Effects of organicacids on the growth and fermentation of ethanologenic Escherichia coliLY01” Biotechnol. Bioengin. 66:203-210).

Oxygen transfer often becomes limiting during aerobic bioconversionprocesses, promoting the accumulation of reduced products (Tsai, P. S.,M. Nageli, and J. E. Bailey, 2002 “Intracellular expression ofVitreoscilla hemoglobin modifies microaerobic Escherichia colimetabolism through elevated concentration and specific activity of thecytochrome o” Biotechnol. Bioeng. 79:558-567; Varma, A., B. W. Boesch,and B. O. Palsson, 1993 “Stoichiometric interpretation of Escherichiacoli glucose catabolism under various oxygenation rates” Appl. Environ.Microbiol. 59:2465-2473). Synthesis of reduced products was eliminatedby mutations in genes (ΔfocApflB ΔfrdCD ΔldhA ΔadhE) encoding the fourmajor fermentation pathways. Excessive oxygen demand and NADH productionwere also reduced by a deletion in succinate dehydrogenase (sucAΔ). Theresulting strain, TC36 (ΔfocApflBΔfrdCD ΔldhA ΔatpFH ΔsucA ΔadhE)metabolizes sugars to acetate with the efficiency of fermentativemetabolism, diverting a minimum of carbon to cell mass (biocatalyst) andCO₂. By replacing the acetate pathway, a variety of alternative oxidizedproducts can be produced using the mutational strategies employed forthe construction of TC36.

Genetically engineered E. coli TC36 can produce acetate in a simpler,single step process using glucose and mineral salts with titres andyields equivalent or higher than current batch processes. Althoughyields for TC36 were lower than those reported for Clostridiumthermoaceticum which contain the Ljungdahl-Wood Pathway (Cheryan, M., S.Parekh, M. Shah and K. Witjitra, 1997 “Production of acetic acid byClostridium thermoaceticum” Adv. Appl. Microbiol. 43:1-33) and maximumtitres with TC36 are lower than can be achieved by ethanol oxidationusing Acetobacter (Berraud, C., 2000 “Production of highly concentratedvinegar in fed-batch culture” Biotechnol. Lett. 22:451-454), acetateproduction rates by TC36 are almost two-fold higher than both andrequired only mineral salts as nutrients.

E. coli TC36 offers a unique set of advantages over currently employedbiocatalysts for the commercial production of acetate: a single stepprocess using sugars as substrates, high rates of acetate production,high acetate yields, simple nutrition (mineral salts), and a robustmetabolism permitting the bioconversion of hexoses, pentoses, and manydissacharides.

Materials and Methods

Bacterial strains and plasmids. Selected E. coli strains and plasmidsare listed in Table 1.

TABLE 1 Strains and plasmids. Strains & Plasmids RelevantCharacteristics Reference Strains W3110 wild type ATCC 27325 TOP10F′lacI^(q) (episome) Invitrogen SE2279 MG1655, pflB ldhA::Tn10 Laboratorycollection (KTS) SZ33 W3110, ldhA::Tn10 Described herein SZ40 W3110,Δ(focA-pflB)::FRT ΔfrdBC Described herein SZ46 W3110, Δ(focA-pflB)::FRTΔfrdBC Described herein ldhA::Tn10 SZ47 W3110, Δ(focA-pflB)::FRT ΔfrdBCΔldhA Described herein TC20 W3110, ΔadhE::FRT-tet-FRT Described hereinTC21 W3110, ΔatpFH::FRT-tet-FRT Described herein TC23 W3110,Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Described herein Δatp(FH)::FRT-tet-FRTTC24 W3110, Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Described hereinΔatp(FH)::FRT TC25 W3110, ΔsucA::FRT-tet-FRT Described herein TC30W3110, Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Described herein Δatp(FH)::FRTΔadhE::FRT-tet-FRT SE1706 ΔfrdBC zid::Tn10 Footnote¹ TC31 W3110,Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Described herein Δatp(FH)::FRT ΔadhE::FRTTC32 W3110, (Succ

), Δ(focA-pflB)::FRT ΔfrdBC Described herein ΔldhA Δatp(FH)::FRTΔadhE::FRT ΔsucA::FRT-tet-FRT TC35 W3110, (Succ⁺), Δ(focA-pflB)::FRTΔfrdBC Described herein ΔldhA Δatp(FH)::FRT ΔadhE::FRTΔsucA::FRT-tet-FRT TC36 W3110, (Succ⁺), Δ(focA-pflB)::FRT ΔfrdBCDescribed herein ΔldhA Δatp(FH)::FRT ΔadhE::FRT ΔsucA::FRT PlasmidspCR2.1-TOPO bla kan, TOPO ™ TA cloning vector Invitrogen pFT-A bla flplow-copy vector containing Footnote² recombinase andtemperature-conditional pSC101 replicon pKD46 bla γ β exo low-copyvector contaiing red Footnote³ recombinase and temperature-conditionalpSC101 replicon pLOI2065 bla, SmaI fragment containing the FRT flankedDescribed herein tet gene pLOI2800 bla kan sucA Described hereinpLOI2801 bla kan sucA::FRT-tet-FRT Described herein pLOI2802 bla kanadhE Described herein pLOI2803 bla kan adhE::FRT-tet-FRT Describedherein pLOI2805 bla kan atpEFH Described herein pLOI2807 bla kanatpFH::FRT-tet-FRT Described herein ¹Ohta, K., D. S. Beall, J. P. Mejia,K. T. Shanmugam, and L. O. Ingram (1991) “Genetic improvement ofEscherichia coli for ethanol production of chromosomal integration ofZymamonas mobilis genes encoding pyruvate decarboxylase and alcoholdehydrogenase II. Appl. Environ. Microbiol. 57: 893-900. ²Posfai, G., M.D. Koob, H. A. Kirkpatrick, and F. C. Blattner. 1997. Versatileinsertion plasmids for targeted genome manipulations in bacteria:Isolation, deletion, and rescue of the pathogenicity island LEE of theEscherichia coli O157:H7 genome. J. Bacteriol. 179: 4426-4428.³Datsenko, K. A. and B. L. Wanner. 2000. One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products. Proc.Natl. Acad. Sci. USA 97: 6640-6645.

Working cultures of E. coli W3110 (ATCC 27325) derivatives weremaintained on a mineral salts medium (per liter: 3.5 g KH₂PO₄; 5.0 gK₂HPO₄; 3.5 g (NH₄)₂HPO₄, 0.25 g MgSO₄

7H₂O, 15 mg CaCl₂

2H₂O, 0.5 mg thiamine, and 1 ml of trace metal stock) containing glucose(2% in plates; 3% in broth) and 1.5% agar. The trace metal stock wasprepared in 0.1 M HCl (per liter: 1.6 g FeCl₃ 0.2 g CoCl₂

6H₂O, 0.1 g CuCl₂, 0.2 g ZnCl₂

4H₂O, 0.2 g NaMoO₄, and 0.05 g H₃BO₃). MOPS (0.1 M, pH 7.1) was added toboth liquid and solid media (autoclaved separately) when needed for pHcontrol, but was not included in medium used for 10-L fermentations.Minimal medium was also prepared using succinate (1 g L⁻¹) and glycerol(1 g L⁻¹) as sole sources of carbon (nonfermentable). Succinate (1 gL⁻¹) was added as a supplement to glucose-minimal medium when needed.During plasmid and strain construction, cultures were grown inLuria-Bertani (LB) broth or on LB plates (1.5% agar) (Sambrook, J. andD. W. Russell, 2001 “Molecular cloning: A laboratory manual” Cold SpringHarbor Press, Cold Spring Harbor, N.Y.). Glucose (2%) was added to LBmedium for all strains containing mutations in (F₁F₀)H⁺-ATP synthase.Antibiotics were included as appropriate (kanamycin, 50 mg L⁻¹;ampicillin, 50 mg L⁻¹; and tetracycline, 12.5 or 6.25 mg L⁻¹). Fusaricacid plates were used to select for loss of tetracycline resistance.

Genetic methods. Standard methods were used for plasmid construction,phage P1 transduction, electroporation, and polymerase chain reaction(PCR) (Miller, J. H., 1992 “A short course in bacterial genetics: Alaboratory manual and handbook for Escherichia coli and relatedbacteria” Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; Sambrook,J. and D. W. Russell, 2001 “Molecular cloning: A laboratory manual” ColdSpring Harbor Press, Cold Spring Harbor, N.Y.). Chromosomal DNA from E.coli W3110 (and derivatives) served as a template to amplify genes usingprimers complementary to coding regions (ORFmers) purchased from theSigma Scientific Company (St. Louis, Mo.).

PCR products were initially cloned into plasmid vector pCR2.1-TOPO.During plasmid constructions, restriction products were converted toblunt ends using either the Klenow fragment of DNA polymerase (5′overhang) or T4 DNA polymerase (3′ overhang) as needed. Integration oflinear DNA was facilitated by using pKD46 (temperature conditional)containing an arabinose-inducible red recombinase (Datsenko, K. A. andB. L. Wanner, 2000 “One-step inactivation of chromosomal genes inEscherichia coli K-12 using PCR products” Proc. Natl. Acad. Sci. USA97:6640-6645). Integrants were selected for tetracycline resistance(6.25 mg L⁻¹) and screened for appropriate antibiotic resistance markersand phenotypic traits. At each step, mutations were verified by analysesof PCR products and fermentation products. FRT-flanked antibioticresistance genes used for selection were deleted using atemperature-conditional plasmid (pFT-A) expressing FLP recombinase froma chlortetracycline-inducible promoter (Martinez-Morales, F., A. G.Borges, A. Martinez, K. T. Shanmugam, and L. O. Ingram, 1999“Chromosomal integration of heterologous DNA in Escherichia coli withprecise removal of markers and replicons during construction” J.Bacteriol. 181:7143-7148; Posfai, G., M. D. Koob, H. A. Kirkpatrick, andF. C. Blattner, 1997 “Versatile insertion plasmids for targeted genomemanipulations in bacteria: Isolation, deletion, and rescue of thepathogenicity island LEE of the Escherichia coli O157:H7 genome” J.Bacteriol. 179:4426-4428).

A removable tetracycline cassette (FRT-tet-FRT) was constructed(pLOI2065) which is analogous to the kanamycin cassette (FRT-kan-FRT) inpKD4 (Datsenko, K. A. and B. L. Wanner, 2000 “One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products” Proc.Natl. Acad. Sci. USA 97:6640-6645). In both cassettes, flanking FRTsites are oriented in the same direction to allow efficient in vivoexcision by FLP recombinanase (Posfai, G., M. D. Koob, H. A.Kirkpatrick, and F. C. Blattner, 1997 “Versatile insertion plasmids fortargeted genome manipulations in bacteria: Isolation, deletion, andrescue of the pathogenicity island LEE of the Escherichia coli O157:H7genome” J. Bacteriol. 179:4426-4428). Plasmid pLOI2065 contains twoEcoRI sites and two SmaI sites for isolation of the FRT-tet-FRTcassette. The sequence for pLOI2065 has been deposited in GenBank(Accession No. AF521666).

Deletion of adhE. To construct an adhE mutant, the coding region (2.68kbp) was amplified by PCR and cloned into pCR2.1-TOPO. The centralregion of adhE (1.06 kbp) was deleted using HincII (2 sites) andreplaced with a 1.7 kbp SmaI fragment from pLOI2065 containing theFRT-tet-FRT cassette to produce pLOI2803. This plasmid was linearized bydigestion with PvuI and ScaI, and served as a template to amplify (adhEprimers) the 3.17 kbp region containing adhE::FRT-tet-FRT. Amplified DNAwas purified and introduced into W3110 by electroporation. Recombinantsfrom double cross-over events were identified by antibiotic markers,confirmed by analysis of PCR and fermentation products. One clone wasselected and designated TC20.

P1 transduction was used to transfer a mutation (frdBC zid::Tn10) fromSE1706 into SZ32, designated SZ35(ΔfocA-pflB::FRT ΔfrdBC zid::Tn10). Thetet gene was removed from SZ35 by fusaric acid selection to produceSZ40(ΔfocA-pflB::FRT ΔfrdBC).

Deletion of pflB. A focA-pflB::FRT mutation was constructed using themethod of Datsenko and Wanner (Datsenko, K. A. and B. L. Wanner, 2000“One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products” Proc. Natl. Acad. Sci. USA 97:6640-6645). Hybridprimers were designed which are complementary to E. coli chromosomalgenes and to the antibiotic cassette (FRT-kan-FRT) in pKD4. The senseprimer(TTACTCCGTATTTGCATAAAAA-CCATGCGAGTTACGGGCCTATAAGTGTAGGCTGGAGCTGCTTC)(SEQ ID NO:1) consisted of an initial 45 bp (bold) corresponding to the−130 to −85 region of foc followed by 20 bp (underlined) correspondingto the primer 1 region of pKD4. The antisense primerTAGATTGAGTGAAGGTACGAGTAATAACGTCCTGCTGC-TGTTCTCATATGAATATCCTCCTTAG) (SEQID NO:2) consisted of an initial 45 bp (bold) of the C-terminal end ofpflB followed by 20 bp (underlined) corresponding to primer 2 region ofpKD4. The FRT-kan-FRT cassette was amplified by PCR using these primersand pKD4 as the template. After purification, amplified DNA waselectroporated into E. coli BW25113 (pKD46). The resultingkanamycin-resistant recombinant, pAH218, contained FRT-kan-FRT in thedeleted region of pflB (46 bp remaining). A phage P1 lysate preparedfrom AH218 (pflB::FRT-kan-FRT) was used to transfer this mutation intoW3110 to produce strain SZ31 (pflB::FRT-kan-FRT). After verifying thismutation by analyses of PCR products, fermentation products, andantibiotic markers, the kan gene was removed from the chromosome by FLPrecombinase using a temperature-conditional helper plasmid (pFT-A).After removal of helper plasmid by growth at 42° C., the resultingkanomycin-sensitive strain (focA-pflB::FRT) was designated SZ32.

Deletion of focA-pflB:FRT, frdBC, ldhA. The ldhA::Tn10 mutation in Ecoli SE2279 was transduced into E. coli W3110 using phage P1 to producestrain SZ33. P1 phage grown on SZ33 was used to transfer this mutationinto SZ40(Δ(focA-pflB)::FRT ΔfrdCD) to produce SZ46.Tetracycline-sensitive derivatives of SZ46 were selected using fusaricacid medium. One clone was designated SZ47 (Δ(focA-pflB)::FRT ΔfrdBCΔldhA). The ΔldhA mutation in SZ47 was confirmed by the absence oflactate in fermentation broth, an inability to grow anaerobically inglucose-minimal media, and by PCR analysis using ldhA ORFmers (1.0 kbpfor the wild type ldhA as compared to 1.1 kbp for SZ47). The slightlylarger size of the amplified product from SZ47 is attributed to remnantsof Tn10.

Deletion of atpFH. The atpEFH coding region of the atpIBEFHAGDC operonwas amplified by PCR using primers (ORFmers, Sigma Scientific, St.Louis, Mo.) complementary to the 5′-end of the atpE gene and the 3′-endof the atpH. The amplified fragment (1.3 kbp) was cloned intopCR2.1-TOPO and one clone selected in which the atpEFH genes wereoriented to permit expression from the lac promoter (pLOI2805; FIG. 2).The atpF gene and 117 nucleotides at the 5′ end of atpH gene wereremoved from pLOI2805 by digestion with HpaI and BstEII(Klenow-treated). This region was replaced with a 1.7 kbp SmaI fragmentfrom pLOI2065 containing the FRT-tet-FRT cassette to produce pLOI2807(FIG. 2). After digestion with ScaI, pLOI2807 served as a template foramplification of the alpEΔ(FH)::FRT-tet-FRT region (2.4 kbp) using the5′ atpE and 3′ atpH primers. Amplified DNA was precipitated, digestedagain with ScaI to disrupt any residual plasmid, and purified by phenolextraction. This DNA was introduced into E. coli W3110(pKD46) byelectroporation while expressing red recombinase. Plasmid pKD46 waseliminated by growth at 42° C. Recombinants (double cross-over) wereidentified using antibiotic markers (tetracycline resistant; sensitiveto ampicillin and kanamycin) and by the inability to grow onsuccinate-minimal plates or glycerol-minimal plates in the absence ofglucose (fermentable carbon source). Integration was further confirmedby PCR analysis using the 5′ atpE primer and the 3′ atpH primer (1.3 kbpfragment for W3110; 2.3 kbp fragment for mutants). One clone wasselected and designated TC21(Δatp(FH)::FRT-tet-FRT.

Phage P1 was used to transduce the Δatp(FH)::FRT-tet-FRT mutation inTC21 to SZ47 and produce TC23. The let gene was removed from TC23 by theFLP recombinase (pFT-A). After elimination of pFT-A by growth at 42° C.,the Δatp(FH)::FRT mutation was further confirmed by PCR analysis usingthe 5′ atpE primer and the 3′ atpH primer (0.8 kbp for deletion and 1.3kbp for SZ47). The resulting strain was designated TC24(ΔfocA-pflB::FRTΔfrdBC ΔldhA ΔatpFH::FRT).

Deletion of adhE. Phage P1 was used to transduce the ΔadhE::FRT-tet-FRTmutation in TC20 to TC24 and produce TC30. Chromosomal integration wasconfirmed by PCR analysis using adhE primers (2.7 kbp for TC24 and 3.2kbp for the ΔadhE::FRT-tet-FRT mutant). The tet gene was deleted fromTC30 by FLP recombinase using pFT-A. After elimination of pFT-A bygrowth at 42° C., a clone was selected and designatedTC31(Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Δatp(FH)::FRT ΔadhE::FRT).

Deletion of part of sucA. The sucA coding region was amplified usingORFmers. The resulting 2.8 kbp PCR product was cloned into pCR2.1-TOPOto produce pLOI2800 (FIG. 2) in which the sucA coding region wasoriented to permit expression from the lac promoter. A 1.1 kbp fragmentwas removed from central region of sucA by digestion of pLOI2800 withSnaBI and AccI (Klenow-treated). This region was replaced with a 1.7 kbpSmaI fragment containing the FRT-tet-FRT cassette from pLOI2065 toproduce pLOI2801 (FIG. 2). Plasmid pLOI2801 was digested with PvuI andScaI and used as a template to amplify the 3.3 kbp region containingsucA::FRT-tet-FRT using sucA ORFmers. Amplified DNA was precipitated,digested with PvuI and ScaI to disrupt any residual circular plasmid,and extracted with phenol. Purified DNA was electroporated into E. coliW3110(pKD46) while expressing red recombinase. Plasmid pKD46 waseliminated by growth at 42° C. Disruption of sucA was confirmed by PCRanalysis using sucA ORFmers (2.8 kbp fragment for wild type and 3.3 kbpfor sucA::FRT-tet-FRT mutants) and designated TC25.

Phage P1 was used to transduce the sucA::FRT-tet-FRT mutation from TC25into TC31. Transfer was verified by PCR analysis (2.8 kbp for wild typesucA and 3.3 kbp for sucA::FRT-tet-FRT mutants) and phenotype (succ⁻).Inactivation of 2-ketoglutarate dehydrogenase (ΔsucA) in this ΔfrdBCbackground resulted in an undesirable auxotrophic requirement forsuccinate (Succ⁻) during growth on glucose-minimal medium. The resultingstrain was designated TC32(Succ⁻, Δ(focA-pflB)::FRT ΔfrdBC ΔldhAΔatp(FH)::FRT ΔadhE::FRT ΔsucA::FRT-tet-FRT).

Elimination of Succ⁻ mutants. Spontaneous Succ⁺ mutants of TC32 werereadily obtained after serial transfers in glucose-minimal brothcontaining decreasing amounts of succinate (4 mM to 0.4 mM) followed byselection on glucose-minimal plates without succinate. Over 170 cloneswere recovered per ml of culture after enrichment, approximately 3% ofviable cells. Ten clones were tested and all grew well in glucoseminimal broth without succinate and produced acetate as the dominantproduct. One was selected (TC35) for deletion of the tet gene using theFLP recombinase. This deletion was confirmed by analysis of PCR productsusing sucA primers (3.3 kbp for TC35 and 1.8 kbp after tet deletion).The resulting strain was designated TC36 (Succ⁺, Δ(focA-pflB)::FRTΔfrdBC ΔldhA Δatp(FH)::FRT ΔadhE::FRT ΔsucA::FRT).

Total ATPase activity was examined in disrupted cell extracts of TC36and W3110 (wild type). The activity in TC36 (0.355 U mg⁻¹ protein) wasequivalent to 71% of the unmodified parent (0.502 U mg⁻¹ protein),confirming that F₁-ATPase was not inactivated by the ΔatpFH::FRTmutation. This is similar to the levels of ATPase reported for an atpHmutant of E. coli which blocked membrane assembly and coupling tooxidative phosphorylation (Sorgen, P. L., T. L. Caviston, R. C. Perry,and B. D. Cain, 1998 “Deletions in the second stalk of F1F0-ATP synthasein Escherichia coli” J. Biol. Chem. 273:27873-27878).

Fermentation. Acetate production was examined in glucose-minimal mediumcontaining 167 mM glucose using a New Brunswick Bioflow 3000 fermentorwith a 10 L working volume (37° C., dual Rushton impellers, 450 rpm).Dissolved oxygen was maintained at 5% of air saturation (unlessotherwise stated) by altering the proportion of N₂ and O₂. Broth wasmaintained at pH 7.0 by the automatic addition of 11.4 M KOH. For fedbatch experiments, additional glucose was added from a sterile 60%stock. Three fed batch regimes were investigated: A. 3% glucoseinitially with the addition of 3% after 12 h (6% total); B. 6% glucoseinitially with the addition of 4% glucose after 16 h (10% total); C. 3%glucose initially with multiple additions to maintain glucose levelsabove 100 mM.

Seed cultures were prepared by inoculating colonies from a fresh plate(48 h) into 3 ml of glucose-minimal medium (13×100 mm tube) containing0.1 M MOPS. After incubation for 14 h (120 rpm rotator), cultures werediluted 400-fold into 1-L baffled flask containing 200 ml of mineralsalts medium (37° C., 280 rpm). When cells reached 1.5-2.2 OD_(550nm),sufficient culture volume was harvested (5000 rpm, 25° C.) to provide aninoculum of 33 mg dry cell weight L⁻¹ in the 10-L working volume.

Broth samples were removed to measure organic acids, residual glucose,and cell mass. Volumetric and specific rates were estimated frommeasured values for glucose and acetate using Prism software (GraphPadSoftware, San Diego, Calif.). A smooth curve was generated with 10points per min (Lowess method) to fit measured results. The firstderivative (acetate or glucose versus time) of each curve served as anestimate of volumetric rate. Specific rates (mmoles L⁻¹ h⁻¹ mg⁻¹ drycell weight) were calculated by dividing volumetric rates by respectivevalues for cell mass.

ATPase. Cells were grown for enzyme assays as described above for seedcultures. Upon reaching 0.75-1.0 OD_(550nm), cultures were chilled onice and harvested by centrifugation (8000×g, 5 min at 4° C.). Cellpellets were washed 5 times with 0.1 M Tris-HCl (pH 7.55), resuspendedin 1 ml of this buffer, and broken using a model W220F ultrasonic celldisrupter (Heat Systems Ultrasonics, Plainview, N.Y., USA). Total ATPaseactivity in disrupted cell preparations was assayed at pH 7.55essentially as described by Evans (Evans, D. J., Jr., 1969 “Membraneadenosine triphosphate of Escherichia coli: activation by calcium ionand inhibition by cations” J. Bacteriol. 100:914-922). Inorganicphosphate was measured by the method of Rathbun and Betlach (Rathbun, W.B., and M. V. Betlach, 1969 “Estimation of enzymatically producedorthophosphate in the presence of cysteine and adenosine triphosphate”Anal. Biochem. 20:436-445). Results represent an average for threecultures of each strain. Specific activity is expressed as μmol P_(i)released min⁻¹ mg⁻¹ protein.

Total ATPase activity was examined in disrupted cell extracts of TC36and W3110 (wild type). The activity in TC36 (0.355 U mg⁻¹ protein) wasequivalent to 71% of the unmodified parent (0.502 U mg⁻¹ protein),confirming that F₁-ATPase was not inactivated by the ΔatpFH::FRTmutation. This is similar to the levels of ATPase reported for an atpHmutant of E. coli which blocked membrane assembly and coupling tooxidative phosphorylation (Sorgen, P. L., T. L. Caviston, R. C. Perry,and B. D. Cain, 1998 “Deletions in the second stalk of F1F0-ATP synthasein Escherichia coli” J. Biol. Chem. 273:27873-27878).

Analyses. Organic acids and glucose concentrations were determined usinga Hewlett Packard HPLC (HP 1090 series II) equipped with a UV monitor(210 nm) and RI detector. Products were separated using a Bio-Rad HPX87H column (10 μl injection) with 4 mM H₂SO₄ as the mobile phase (0.4 mlmin⁻¹, 45° C.). Cell mass was estimated by measuring OD_(550nm) (1.0OD_(550nm) is equivalent to 0.33 g L⁻¹ dry cell weight) using a Bausch &Lomb Spectronic 70 spectrophotometer with 10×75 mm culture tubes ascuvettes. Protein concentration was determined using the BCA ProteinAssay Kit from Pierce (Rockford, Ill.).

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Construction of a Homo-Acetate Fermentation Pathway in E. coliW3110

Fermentation of sugars through native pathways in E. coli produces amixture of organic acids, ethanol, CO₂ and H₂ (FIG. 1). Acetate andethanol are typically produced in approximately equimolar amounts fromacetyl˜CoA to provide redox balance (Clark, D. P., 1989 “Thefermentation pathways of Escherichia coli. FEMS” Microbiol. Rev.63:223-234; de Graef, M. R., S. Alexeeva, J. L. Snoep, and M. J.Teixiera de Mattos, 1999 “The steady-state internal redox state(NADH/NAD) reflects the external redox state and is correlated withcatabolic adaptation in Escherichia coli” J. Bacteriol. 181:2351-2357).To construct a strain for homo-acetate production, removable antibioticresistance genes were used to sequentially inactivate chromosomal genesencoding alternative pathways.

Inspection of native pathways in E. coli (FIG. 1) indicated that theproduction of acetate and CO₂ as sole metabolic products from glucosewill require an external electron acceptor such as oxygen. Due to lowoxygen solubility, however, it is difficult to satisfy the oxygen demandfrom active E. coli metabolism and a portion of substrate is typicallyconverted to fermentation products such as lactate and ethanol. Thisproblem was eliminated by combining deletions in genes encoding lactatedehydrogenase, pyruvate formatelyase, and alcohol/aldehydedehydrogenase.

A deletion was inserted into the pflB gene, the ldhA gene, and the adhEgene of W3110. These mutations eliminated the production of CO₂,lactate, and ethanol in 3% glucose-minimal media (Table 2).

TABLE 2 Comparison of metabolic rates. Max Vol^(a) Max Spec Max Vol^(a)Max Spec^(b) Glucose Glucose^(b) Acetate Acetate Specific UtilizationUtilization Production Production Growth (mmol (mmol (mmol (mmol StrainRate (μ) liter⁻¹ h⁻¹) h⁻¹ g⁻¹) liter⁻¹ h⁻¹) h⁻¹ g⁻¹) W3110 0.87 18 9 9.510 SZ47 0.87 22 11 9 10 TC24 0.78 28 20 26 16 TC36 0.69 33 18 23 16^(a)Maximum volumetric rates for glucose utilization and acetateproduction. ^(b)Maximim specific rates (dry cell weight basis) forglucose utilization and acetate production. Values for glucoserepresents a measure of maximal glycolytic flux.

Several different mutations can be used to block succinate production(FIG. 1). During fermentation, the tricarboxlyic acid (TCA) pathwayserves primarily as a source of carbon skeletons for biosynthesis.Previous experience with E. coli B strains (Ingram, L. O., H. C.Aldrich, A. C. C. Borges, T. B. Causey, A. Martinez, F. Morales, A.Saleh, S. A. Underwood, L. P. Yomano, S. W. York, J. Zaldivar, and S.Zhou, 1999 “Enteric bacterial catalysts for fuel ethanol production.”Biotechnol. Prog. 15:855-866) engineered for ethanol production hasshown that a deletion in the frdABCD operon can be used as analternative method to block succinate production by preventing theproduction of fumarate reductase. Thus, the deletion of the frdCD geneeliminates the production of succinate by reductive reactions.

The TCA cycle was further disrupted by the deletion of sucA (FIG. 1)During oxidative growth, up to 50% of substrate carbon can be lost asCO₂ (Neidhardt, F. C., J. L. Ingraham, and M. Schaechter, 1990“Physiology of the bacterial cell: A molecular approach” SinauerAssociates, Inc., Sunderland, Mass.). This loss of carbon can beattributed in large measure to the high efficiency of the TCA cycle andthe electron transport system (NADH oxidation). During fermentativemetabolism, the production of CO₂ and NADH are reduced primarily bystrong repression of sucAB encoding 2-ketoglutarate dehydrogenase(Cunningham, L. and J. R. Guest, 1998 “Transcription and transcriptprocessing in the sdhCDAB-sucABCD operon of Escherichia coli”Microbiology 144:2113-2123; Park, S.-J., G. Chao, and R. P. Gunsalus,1997 “Aerobic regulation of the sucABCD gene of Escherichia coli, whichencode α-ketoglutarate dehydrogenase and succinyl coenzyme A synthetase:roles of ArcA, Fnr, and the upstream sdhCDAB promoter” J. Bacteriol.179:4138-4142), disrupting the cyclic function of the TCA cycle.Deleting part of the sucA gene imposed a restriction in carbon flowthrough the TCA cycle.

Again, growth under oxidative conditions is characterized by conversionof up to 50% of substrate carbon to cell mass (Neidhardt, F. C., J. L.Ingraham, and M. Schaechter, 1990 “Physiology of the bacterial cell: Amolecular approach” Sinauer Associates, Inc., Sunderland, Mass.). Toreduce the potential drain of substrate into cell mass, a mutation wasintroduced into SZ47 that deleted portions of two subunits in(F₁F₀)H⁺-ATP synthase concerned with assembly to the plasma membrane(Sorgen, P. L., T. L. Caviston, R. C. Perry, and B. D. Cain, 1998“Deletions in the second stalk of F1F0-ATP synthase in Escherichia coli”J. Biol. Chem. 273:27873-27878), disrupting oxidative phosphorylationwhile preserving the hydrolytic activity of F₁-ATPase in the cytoplasm.Thus, the strain is able to grow in minimal medium without a fermentablecarbon source (substrate level phosphorylation) and retains the abilityto oxidize NADH by the electron transport system.

These deletions resulted in strain TC32, which required succinate forgrowth on glucose-minimal medium. Thus, spontaneous Succ⁺ mutants ofTC32 were obtained by performing serial transfers in glucose-minimalbroth containing decreasing amounts of succinate followed by selectionon glucose-minimal plates without succinate.

The resulting strain, TC36 has absolute requirements for a fermentablecarbon source (substrate level phosphoylation) and for an externalelectron acceptor that can couple to the electron transport systemduring growth in mineral salts medium to maintain redox balance. Withgenetic blocks in all major fermentation pathways and oxidativephosphorylation, this strain is relatively insensitive to variations indissolved oxygen. TC36(ΔfocApflB ΔfrdCD ΔldhA ΔatpFH ΔsucA ΔadhE)metabolizes sugars to acetate with the efficiency of fermentativemetabolism, diverting a minimum of carbon to cell mass (biocatalyst) andCO₂. By replacing the acetate pathway, a variety of alternative oxidizedproducts can be produced using the mutational strategies employed forthe construction of TC36.

EXAMPLE 2 Effects of Gene Disruptions on Growth and Glycolytic Flux

TC36 was genetically engineered for the production of acetate fromcarbohydrates such as glucose. Batch fermentations with pH control wereused to compare the performance of this strain with W3110 (wild type)and two intermediate strains used for construction, SZ47(ΔpflB, ΔfrdCD,ΔldhA) and TC24(ΔpflB, ΔfrdCD, ΔldhA ΔatpFH). Under 5% oxygen saturationand 3% glucose (37° C.) test conditions, the broth pH was maintained atneutrality to minimize toxicity from undissociated acids (Chotani, G.,T. Dodge, A. Hsu, M. Kumar, R. LaDuca, D. Trimbur, W. Weyler, and K.Sanford, 2000 “The commercial production of chemicals using pathwayengineering” Biochim. Biophys. Acta 1543:434-455).

Disruption of oxidative phosphorylation and the cyclic function of thetricarboxylic acid cycle, elimination of the primary fermentationpathways, and the production of acetate as the primary end-product fromglycolysis had relatively little effect on the growth of E. coli Themaximum growth rates for strains W3110 (wild type) and SZ47 (lacking thethree native fermentation pathways) were similar although the cell yieldfor SZ47 was higher (FIG. 3A; Table 2 and Table 3). Inactivation ofoxidative phosphorylation (ΔatpFH) resulted in a small reduction ingrowth rate and cell yield (TC24). Cell yield and growth rate werelowest for strain TC36 containing additional mutations in2-ketoglutarate dehydrogenase (ΔsucA) and alcohol dehydrogenase (ΔadhE),approximately 80% of the unmodified parent W3110.

Maximal rates for glucose utilization (specific and volumetric) werehigher for TC36 and TC24 than for W3110 and SZ47 (Table 2). Thisincrease in metabolic activity can be primarily attributed to the ΔatpFHmutation. ATP levels serve as an allosteric regulator of several keyglycolytic enzymes (Neidhardt, F. C., J. L. Ingraham, and M. Schaechter,1990 “Physiology of the bacterial cell: A molecular approach” SinauerAssociates, Inc., Sunderland, Mass.), and acetate kinase (Suzuki, T.,1969 “Phosphotransacetylase of Escherichia coli B, activation bypyruvate and inhibition by NADH and certain nucleotides” Biochim.Biophys. Acta 191:559-569). Differences between strains wereparticularly evident when comparing incubation times required tocomplete sugar metabolism (FIG. 3B). With TC36 and TC24, glucose wasexhausted in 16-18 h as compared to 26 h for SZ47 and 30 h for W3110.The maximum specific rate of glucose utilization (glycolytic flux) was 9mmole h⁻¹ g⁻¹ dry cell weight in the unmodified parent (W3110), 20 mmoleh⁻¹ g⁻¹ dry cell weight in TC24, and 18 mmole h⁻¹ g⁻¹ dry cell weight inTC36. The slightly lower glycolytic flux in TC36 as compared to TC24 maybe related to the increase in ATP yield resulting from improvements inacetate yield (1 ATP per acetate). Assuming protein represents 55% ofdry cell weight, maximal glycolytic flux in TC36 is approximately 0.55μmoles glucose min⁻¹ mg⁻¹ protein.

The (F₁F₀)H⁺-ATP synthase and 2-ketoglutarate dehydrogenase mutationsintroduced into TC36 to minimize the levels of ATP and NAD(P)H fromglucose under oxidative conditions also promote glycolysis throughnative allosteric controls (Neidhardt, F. C., J. L. Ingraham, and M.Schaechter, 1990 “Physiology of the bacterial cell: A molecularapproach” Sinauer Associates, Inc., Sunderland, Mass.; Underwood, S. A.,M. L. Buszko, K. T. Shanmugam, and L. O. Ingram, 2002 “Flux throughcitrate synthase limits the growth of ethanologenic Escherichia coliKO11 during xylose fermentation” Appl. Environ. Microbiol.68:1071-1081), providing a mechanism for the observed 2-fold increase inglycolytic flux as compared to W3110(wild type).

EXAMPLE 3 Production of Other Organic Acids

A substantial portion of glucose carbon was not recovered in the carbonbalance (Table 3) for W3110 (40%) and SZ47 (80%). This loss isattributed to the production of volatile products by high flux throughthe tricarboxylic acid cycle (CO₂) but may also include the reduction ofacetyl˜CoA to acetaldehyde and ethanol (FIG. 1).

TABLE 3 Summary of fermentation products. Cell Carbon Yield FermentationProducts^(a) (mM) Yield^(b) Recovery^(c) Strain Conditions (g/liter)Acetate 2-ketoglutarate Fumaratc Lactatc Pyruvate Succinate (%) (%substrate C) W3110 3% glucose 4.5 30 39 0.8 33 <1 5 9 60 5% DO SZ47 3%glucose 5.3 6 11 0.9 <1 1 3 2 20 5% DO TC24 3% glucose 4.4 156 1 1.0 <1<1 2 47 66 5% DO TC36 3% glucose 3.5_0.2 224_14 16_6 0.4_0.1 <1 0_0.54_1 68 89 5% DO TC 36 3% glucose 3.2 190 24 <1 <1 <1 3 57 88 15% DO TC363% Glucose 2.5 220 31 <1 <1 <1 10 66 95 5% DO N-limited TC36 3 + 3%glucose 3.8 523 21 <1 3 14 2 78 95 5% DO TC36 3 + 3% glucose 3.0 572 33<1 <1 <1 6 86 102 5% DO N-limited TC36 6% glucose 4.18 415 47 0.3 <1 467 62 92 5% DO TC36 6 + 4% glucose^(d) 4.5 767 37 0.5 <1 72 5 72 97 5% DOTC36 Fed batch^(e) 4.1 878 33 3.4 <1 <1 25 75 88 5% DO^(a)Concentrations in broth after all glucose had been depleted, exceptas noted. ^(b)Yield expressed as a percentage of the maximal theoreticalyield (0.67 g acetate per g glucose). ^(c)Carbon recovery represents thepercentage of substrate carbon recovered. Recovered carbon wascalculated as the sum of carbon in cell mass, fermentation products, andCO2. ^(d)In the final sample, 44 mM glucose was present. ^(e) Excessglucose (9.5%) was added to fermentation to maintain levels above 100mM; 107 mM glucose was present in the final sample.

Although ethanol was absent in broth samples from all pH-controlledfermentations (sparged at 1 L min⁻¹), a small amount of ethanol (6 mM)was found in seed cultures of W3110 (shaken flasks). No ethanol waspresent in seed cultures of TC36, because of the mutation in alcoholdehydrogenase E (adhE). In W3110, the electron transport system (5%dissolved oxygen) and native fermentation pathways (Table 3) serve ascomplementary routes for NADH oxidation.

Eliminating the fermentation pathways to produce the strain SZ47,doubled the loss of carbon as volatile products (Table 3) through theTCA cycle. While SZ47 cell yield increased, the rate of acetateproduction in comparison to W3110 decreased (Table 2 and Table 3).

Strain W3110 accumulated the highest levels of dicarboxylic acids(primarily succinate and 2-ketoglutarate produced through the TCA cycle)during glucose metabolism, approximately 3-fold that of the engineeredstrains (FIG. 4B). The order of appearance of dicarboxylic acids in thebroth correlated with growth rate and the order in which each strainentered into stationary phase. Dicarboxylic acids were partiallyconsumed as glucose levels declined, and may represent spilloverproducts from excessive glycolysis during the transition fromexponential to stationary phase. Although dicarboxylic acids wereproduced by each strain, no significant accumulation of pyruvate wasobserved for W3110, SZ47 or TC24.

Pyruvate levels in the broth of TC36 increased (16 mM at 12 h) duringthe transition stage (FIG. 4C). Although this pyruvate was subsequentlymetabolized, the excretion of pyruvate indicates that glucose uptake andglycolysis per se may not be limiting for acetate production. Because ofthe various mutations in TC36, metabolism of pyruvate is limitedprimarily to small biosynthetic needs and conversion to acetyl˜CoA bythe pyruvate dehydrogenase complex (FIG. 1). Although pyruvatedehydrogenase is activated by low NADH, acetyl˜CoA production may belimited by the availability of free CoA. Resulting rises in pyruvatepools (FIG. 4C), would serve as an allosteric activator ofphosphotransferase (Suzuki, T., 1969 “Phosphotransacetylase ofEscherichia coli B, activation by pyruvate and inhibition by NADH andcertain nucleotides” Biochim. Biophys. Acta 191:559-569), sincephosphotransferase (pta) is the first committed step for acetateproduction from acetyl˜CoA (FIG. 1). Gratuitous ATP hydrolysis byF1-ATPase (FIG. 1C) should ensure the availability of ADP for the finalstep in acetate production catalyzed by acetate kinase (ackA) (FIG. 1).Excess pyruvate can also be directly oxidized to acetate by pyruvateoxidase (poxB), an enzyme that is induced during the latter stages ofgrowth and by environmental stress (Chang, Y.-Y., A.-Y. Wang, and J. E.Cronan, Jr., 1994 “Expression of Escherichia coli pyruvate oxidase(PoxB) depends on the sigma factor enocoded by the rpoS (katF) gene”Mol. Microbiol. 11:1019-1028). Thus, pyruvate oxidase (poxB) may alsocontribute to acetate production by TC36.

Total organic acid production can be measured by the consumption of baseto maintain pH 7.0 (FIG. 3C). Consistent with a more rapid glucosemetabolism, TC24 and TC36 exhibit higher rates and maxima. In general,variations in glucose utilization were accompanied by correspondingchanges in base utilization. Thus, a higher consumption of basecorresponds to a higher utilization of glucose. The exponential natureof the early time points reflects growth of the biocatalysts.

EXAMPLE 4 Production of Acetate

Inactivation of oxidative phosphorylation (ΔatpFH) in SZ47 to produceTC24 resulted in a 5-fold increase in acetate yield and a 3-foldimprovement in carbon recovery, (Table 3), since less carbon was used inthe production of cell mass. Acetate yield and carbon recovery increasedby another 30% with the introduction of the sucA and adhE mutations toproduce TC36. The sucA mutation disrupted the TCA cycle, while the adhEmutation blocked the production of ethanol; therefore, both mutationsdirected carbon atoms to the production of acetate instead of othercompeting products. With 3% glucose mineral salts medium, TC36 producedan average of 224 mM acetate in 16 h with only small amounts of othercompeting products (Table 2). This represents 68% of the maximumtheoretical yield using native pathways (2 acetates per glucose),remaining carbon being divided between cell mass, dicarboxylic acids,and CO₂.

The maximal rates of acetate production (specific and volumetric) wereapproximately 2-fold higher for TC24 and TC36 than for SZ47 and W3110(Table 3), a difference which can be attributed solely to the mutationin the (F₁F₀)H⁺-ATP synthase. This mutation eliminated ATP production byoxidative phosphorylation while retaining cytoplasmic (F₁F₀)H⁺-ATPsynthase for the gratuitous consumption of ATP. Thus, less carbon wasused in building cell mass, but rather carbon was efficiently directedto the assimilation of acetate.

The consumption of base to maintain pH 7.0 provides an overall measureof total organic acid production (FIG. 3C). Higher rates and maxima forTC24 and TC36 are consistent with more rapid glucose metabolism. Ingeneral, variations in glucose utilization were accompanied bycorresponding changes in base utilization. Thus, a higher consumption ofbase corresponds to a higher utilization of glucose. The exponentialnature of the early time points reflects growth of the biocatalysts.

EXAMPLE 5 Improving Acetate Yields

Dicarboxylic acids and cell mass were the dominant competing co-productsfrom glucose. In order to evaluate the potential for process changes toimprove acetate yield, experiments were conducted. Acetate yield was notimproved by increasing the oxygen level from 5% dissolved oxygen to 15%dissolved oxygen, by reducing ammonia nitrogen (2 g L⁻¹ ammoniumphosphate) by 40% to limit growth, or by increasing the initialconcentration of glucose from 3% to 6% (Table 3).

However, a simple two-step batch feeding strategy was beneficial. Asecond addition of 3% glucose at the end of the growth phase (12 h) wasmetabolized to completion and produced 523 mM acetate with minimalincrease in cell mass (FIG. 5). Acetate yield for this two-step addition(6% total glucose) was 78% of the theoretical maximum as compared to 68%for 3% glucose. The highest acetate yield, 86% of the theoreticalmaximum, was obtained by combining the one-step addition of 3% glucosewith the nitrogen limitation (Table 3). Additional fed-batch experimentswere conducted in which multiple additions were made to glucose levelsabove 100 mM. With this approach, 878 mM acetate was producedrepresenting 75% of the maximum theoretical yield (Table 3).

Strain TC36 can be used as a biocatalysis platform for the efficientproduction of oxidized products. Under conditions of glucose excess,strain TC36 produced a maximum of 878 mM acetate, 75% of the maximumtheoretical yield (Table 3) or 0.50 g acetate per g glucose. Along withthe acetate, only cell mass and small amounts of organic acids wereproduced. It is likely that 878 mM acetate approaches the upper limit oftolerance for the metabolism in TC36.

Concentrations as low as 50 mM acetate have been shown to induce astress response in E. coli (Kirkpatrick, C., L. M. Maurer, N. E.Oyelakin, Y. N. Yoncheva, R. Maurer, and J. L. Slonczewski, 2001“Acetate and formate stress: Opposite responses in the proteomes ofEscherichia coli” J. Bacteriol. 183:6466-6477). The minimal inhibitoryconcentration for growth has been previously reported as 300-400 mMacetate at neutral pH (Lasko, D. R., N. Zamboni, and U. Sauer, 2000“Bacterial response to acetate challenge: a comparison of toleranceamong species” Appl. Microbiol Biotechnol. 54:243-247; Zaldivar, J., andL. O. Ingram, 1999 “Effects of organic acids on the growth andfermentation of ethanologenic Escherichia coli LY01” Biotechnol.Bioengin. 66:203-210). Oxygen transfer often becomes limiting duringaerobic bioconversion processes, promoting the accumulation of reducedproducts (Tsai, P. S., M. Nageli, and J. E. Bailey, 2002 “Intracellularexpression of Vitreoscilla hemoglobin modifies microaerobic Escherichiacoli metabolism through elevated concentration and specific activity ofthe cytochrome o” Biotechnol. Bioeng. 79:558-567; Varma, A., B. W.Boesch, and B. O. Palsson, 1993 “Stoichiometric interpretation ofEscherichia coli glucose catabolism under various oxygenation rates”Appl. Environ. Microbiol. 59:2465-2473).

Synthesis of reduced products was eliminated by mutations in genes(ΔfocApflB ΔfrdCD ΔldhA ΔadhE) encoding the four major fermentationpathways. Excessive oxygen demand and NADH production were also reducedby a deletion in succinate dehydrogenase (sucAΔ). The resulting strain,TC36(ΔfocApflBΔfrdCD ΔldhA ΔatpFH ΔsucA ΔadhE) metabolizes sugars toacetate with the efficiency of fermentative metabolism, diverting aminimum of carbon to cell mass (biocatalyst) and CO₂. By replacing theacetate pathway, a variety of alternative oxidized products can beproduced using the mutational strategies employed for the constructionof TC36.

E. coli TC36 offers a unique set of advantages over currently employedbiocatalysts for the commercial production of acetate: a single stepprocess using sugars as substrates, high rates of acetate production,high acetate yields, simple nutrition (mineral salts), and a robustmetabolism permitting the bioconversion of hexoses, pentoses, and manydissacharides.

EXAMPLE 6 Production of Pyruvic Acid Materials and Methods

Microorganisms and media. Strains and plasmids used according to thisExample 6 are listed in Table 4. Working cultures of E. coli W3110 (ATCC27325) and derivatives were maintained on a minimal medium containingmineral salts (per liter: 3.5 g KH₂PO₄; 5.0 g K₂HPO₄; 3.5 g (NH₄)₂HPO₄,0.25 g MgSO₄.7H₂O, 15 mg CaCl₂.2H₂O, 0.5 mg thiamine, and 1 ml of tracemetal stock), glucose (2% in plates; 3% in broth), and 1.5% agar. Thetrace metal stock was prepared in 0.1 M HCl (per liter: 1.6 g FeCl₃, 0.2g CoCl₂.6H₂O, 0.1 g CuCl₂, 0.2 g ZnCl₂.4H₂O, 0.2 g NaMoO₄, and 0.05 gH₃BO₃). MOPS (0.1 M, pH 7.4) was added to both liquid and solid mediawhen needed for pH control, but was not included in pH-controlledfermentations. During plasmid and strain construction, cultures weregrown in Luria-Bertani (LB) broth or on LB plates (1.5% agar) (Miller,J. H. 1992). Glucose (2%) was added to LB medium for all strainscontaining mutations in (F₁F₀)H⁺-ATP synthase. Antibiotics were includedas appropriate (kanamycin, 50 mg L⁻¹; ampicillin, 50 mg L⁻¹; apramycin,50 mg L⁻¹; and tetracycline, 12.5 or 6.25 mg L⁻¹).

Genetic Methods. Standard methods were used for plasmid construction,phage P1 transduction, electroporation, and polymerase chain reaction(PCR) (Miller, J. H., 1992 “A short course in bacterial genetics: Alaboratory manual and handbook for Escherichia coli and relatedbacteria. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; Sambrook,J. and D. W. Russell, 2001 Molecular cloning: A laboratory manual. ColdSpring Harbor Press, Cold Spring Harbor, N.Y.). Chromosomal DNA servedas a template to amplify ackA and poxB genes using primers (ORFmers)complementary to coding regions purchased from Sigma-Genosys, Inc. (TheWoodlands, Tex.). PCR products were initially cloned into plasmid vectorpCR2.1-TOPO. Integration of linear DNA was facilitated by using pKD46(temperature conditional) containing an arabinose-inducible Redrecombinase (Datsenko, K. A. & Wanner, B. L. 2000). Integrants wereselected for tetracycline (6.25 mg L⁻¹) resistance and screened forappropriate antibiotic resistance markers and phenotypic traits. At eachstep, mutations were verified by analyses of PCR products andfermentation profiles. The FRT-flanked antibiotic resistance genes usedfor selection were deleted using a temperature-conditional plasmid(pFT-A) expressing FLP recombinase from a chlortetracycline-induciblepromoter (Martinez-Morales, F., A. G. Borges, A. Martinez, K. T.Shanmugam, and L. O. Ingram, 1999 “Chromosomal integration ofheterologous DNA in Escherichia coli with precise removal of markers andreplicons during construction” J. Bacteriol. 181:7143-7148; Posfai, G.,M. D. Koob, H. A. Kirkpatrick, and F. C. Blattner, 1997 “Versatileinsertion plasmids for targeted genome manipulations in bacteria:Isolation, deletion, and rescue of the pathogenicity island LEE of theEscherichia coli O157:H7 genome” J. Bacteriol. 179:4426-4428).

Disruption of pyruvate oxidase (poxB). The poxB coding region (1.7 kbp)was amplified by PCR using primers (ORFmers) obtained from Sigma-Genosys(The Woodlands, Tex.) and ligated into pCR2.1-TOPO. A single clone wasselected in which the poxB gene was oriented in the same direction asthe lac promoter (pLOI2075). To eliminate extraneous BsaBI sites in thevector, the EcoRI fragment from pLOI2075 containing poxB was ligatedinto the unique EcoR1 site of pLOI2403 to produce plasmid pLOI2078. Thesmall SmaI fragment (1.63 kbp) from pLOI2065 containing a tet geneflanked by FRT sites was ligated into the unique BsaBI site within thepoxB gene in pLOI2078 to produce pLOI2080. After digestion with HindIII,pLOI2080 served as a template for the amplification of poxB::FRT-tet-FRT(3.4 kbp) using poxB primers. Amplified DNA was electroporated into E.coli W3110(pKD46) while expressing Red recombinase. Plasmid pKD46 waseliminated by incubation at 42° C. Double crossover recombinants wereidentified using antibiotic markers (tetracycline resistant; sensitiveto ampicillin and kanamycin) and confirmed by PCR analysis using thepoxB ORFmers (1.7 kbp fragment for W310; 3.4 kbp fragment for mutants).One clone was selected and designated LY74.

Phage P1 was used to transduce the poxB::FRT-tet-FRT mutation from LY74into TC36 to produce TC41. The tet gene was removed from TC41 using theFLP recombinase (pFT-A). After elimination of pFT-A by growth at 42° C.,the poxB::FRT was confirmed by a comparison of PCR products using poxBprimers (1.8 kbp for the mutant; 1.7 kbp for the wild type). Theresulting strain was designated TC42 [(focA-pflB::FRT) frdBC::FRT ldhAatpFH::FRT adhE: FRT sucA::FRT poxB::FRT].

TABLE 4 Sources and characteristics of strains and plasmids used inExample 6. Strains/ Plasmids Relevant Characteristics Reference StrainsW3110 K12 wild type ATCC 27325 TOP10F′ lacI^(q) (episome) InvitrogenLY01 E. coli B, frd pfl::pdc_(Zm) adhE_(Zm) cat Footnote¹ LY74 W3110,ΔpoxB::FRT-tet-FRT Described herein SZ61 W3110, ΔackA::FRT-tet-FRTFootnote² TC36 W3110, (Succ⁺), Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Footnote³Δatp(FH)::FRT ΔadhE::FRT ΔsucA::FRT TC37 W3110, (Succ⁺),Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Described herein Δatp(FH)::FRT ΔadhE::FRTΔsucA::FRT ΔackA::FRT- tet-FRT TC38 W3110, (Succ⁺), Δ(focA-pflB)::FRTΔfrdBC ΔldhA Described herein Δatp(FH)::FRT ΔadhE::FRT ΔsucA::FRTΔackA::FRT TC41 W3110, (Succ⁺), Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Describedherein Δatp(FH)::FRT ΔadhE::FRT ΔsucA::FRT ΔpoxB::FRT- tet-FRT TC42W3110, (Succ⁺), Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Described hereinΔatp(FH)::FRT ΔadhE::FRT ΔsucA::FRT ΔpoxB::FRT TC43 W3110, (Succ⁺),Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Described herein Δatp(FH)::FRT ΔadhE::FRTΔsucA::FRT ΔpoxB::FRT ΔackA::FRT-tet-FRT TC44 W3110, (Succ⁺),Δ(focA-pflB)::FRT ΔfrdBC ΔldhA Described herein Δatp(FH)::FRT ΔadhE::FRTΔsucA::FRT ΔpoxB::FRT ΔackA::FRT Plasmids pCR2.1- bla kan, TOPO ™ TAcloning vector Invitrogen TOPO pFT-A bla flp low-copy vector containingrecombinase and Footnote⁴ temperature-conditional pSC101 replicon pKD46bla γ β exo low-copy vector containing red recombinase Footnote⁵ andtemperature-conditional pSC101 replicon pLOI2065 bla, SmaI fragment withFRT flanked tet gene Footnote⁶ pLOI2075 bla kan poxB Described hereinpLOI2078 bla poxB Described herein pLOI2080 bla poxB::FRT-tet-FRTDescribed herein pLOI2403 bla Footnote⁷ ¹Yomano, L. P., S. W. York, andL. O. Ingram. 1998. Isolation and characterization of ethanol-tolerantmutants of Escherichia coli KO11 for fuel ethanol production. J. Ind.Microbiol. Biot. 20: 132-138. ²Zhou, S., T. B. Causey, A. Hasona, K. T.Shanmugam and L. O. Ingram. 2003. Production of optically pure D-lacticacid in mineral salts medium by metabolically engineered Escherichiacoli W3110. Appl. Environ. Microbiol. 69: 399-407. ³Causey, T. B., S.Zhou, K. T. Shanmugam, L. O. Ingram. 2003. Engineering Escherichia coliW3110 for the conversion of sugar to redox-neutral and oxidizedproducts: Homoacetate production. Proc. Natl. Acad. Sci, USA. 100:825-832. ⁴Posfai, G., M. D. Koob, H. A. Kirkpatrick, and F. C. Blattner.1997. Versatile insertion plasmids for targeted genome manipulations inbacteria: Isolation, deletion, and rescue of the pathogenicity islandLEE of the Escherichia coli O157:H7 genome. J. Bacteriol. ⁵Datsenko, K.A. and B. L. Wanner. 2000. One-step inactivation of chromosomal genes inEscherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645. ⁶Underwood, S. A., S. Zhou, T. B. Causey, L. P. Yomano, K. T.Shanmugam, and L. O. Ingram. 2002. Genetic changes to optimize carbonpartitioning between ethanol and biosynthesis in ethanologenicEscherichia coli. Appl. Environ, Microbiol. 68: 6263-6272.⁷Martinez-Morales, F., A. G. Borges, A, Martinez, K. T. Shanmugam, andL. O. Ingram. 1999. Chromosomal integration of heterologous DNA inEscherichia coli with precise removal of markers and replicons duringconstruction. J. Bacteriol. 181: 7143-7148.

Deletion of ackA (acetate kinase). Phage P1 was used to transduce theackA::FRT-tet-FRT mutation from SZ61 (Zhou, S., T. B. Causey, A. Hasona,K. T. Shanmugam and L. O. Ingram, 2003 “Production of optically pureD-lactic acid in mineral salts medium by metabolically engineeredEscherichia coli W3110” Appl. Environ. Microbiol. 69:399-407) into TC36and TC42 to produce strain TC37 [(focA-pflB::FRT)frdBC::FRT.ldhA.atpFH::FRT.adhE::FRT.sucA::FRT.ackA::FRT-tet-FRT] andTC43 [(focA-pflB::FRT) frdBC::FRT.ldhA.atpFH::FRT.adhE::FRT.sucA::FRTpoxB::FRT.ackA::FRT-tet-FRT], respectively. Chromosomal integration wasverified by comparison of PCR products obtained from SZ61(2.8 kbp) andW3110 (1.2 kbp) using ackA primers (ORFmers, Sigma-Genosys). A reductionin acetate production was verified for each strain by HPLC analysis ofbroth obtained from overnight cultures grown in mineral salts mediumcontaining 167 mM glucose (37° C., 120-rpm). Plasmid pFT-A containingthe FLP recombinase was used to excise the tet genes. After removal ofthis plasmid by incubation at 42° C., resulting strains were designatedTC38 [(focA-pflB::FRT)frdBC::FRT.ldhA.atpFH::FRT.adhE::FRT.sucA::FRT.ackA::FRT) and TC44[(focA-pflB::FRT)frdBC::FRT.ldhA.atpFH::FRT.adhE::FRT.sucA::FRTpoxB::FRT.ackA::FRT], respectively.

Fermentation. Ten-liter batch fermentations (37° C., dual Rushtonimpellers, 450 rpm) with strain TC36 were conducted in minimal mediumcontaining glucose (170 mM and 340 mM) using New Brunswick Bioflow 3000fermentors (New Brunswick Scientific) as described previously (Causey,T. B., S. Zhou, K. T. Shanmugam, L. O. Ingram, 2003 “EngineeringEscherichia coli W3110 for the conversion of sugar to redox-neutral andoxidized products: Homoacetate production” Proc. Natl. Acad. Sci, USA100:825-832). Five-liter batch fermentations (37° C., dual Rushtonimpellers, 350 rpm) were carried out in 8 L vessels. Unless statedotherwise, dissolved oxygen levels were 100% of air saturation at thetime of inoculation and allowed to fall to 5% of air saturation duringcontinuous sparging with air (0.2 vvm). This 5% level was maintainedduring subsequent incubation by mixing O₂ with air while maintaining aconstant flow rate of 10.0 L min⁻¹. Broth was maintained at pH 7.0 bythe automatic addition of 11.4 M KOH. During fed-batch experiments,glucose was added from a sterile 4 M stock. Two fed batch regimes wereinvestigated: 1) 3% initial glucose followed the addition of 3% glucoseafter 15 h (6% total); 2) 3% initial glucose with the addition of 590 mlof 4 M glucose at a constant rate over a 20-h period (9.8% totalglucose).

Seed cultures were prepared by inoculating colonies from a fresh plate(48 h) into 3 ml of glucose-minimal medium (13×100 mm tube) containing0.1 M MOPS. One ml of this cell suspension was diluted 100-fold into 1-Lbaffled flasks containing 200 ml of mineral salts medium (37° C., 280rpm). When cells reached 1.0-1.5 OD_(550nm), sufficient culture volumewas harvested (5000×g, 25° C.) to provide an inoculum of 16.5 mg drycell weight L⁻¹.

Broth samples were removed to measure organic acids, residual glucose,and cell mass. Volumetric and specific rates were estimated frommeasured values for glucose and acetate using GraphPad Prism (GraphPadSoftware, San Diego, Calif.). A smooth curve was generated with 10points per min (Lowess method) to fit measured results. The firstderivative (acetate or glucose versus time) of each curve served as anestimate of volumetric rate. Specific rates (mmoles L⁻¹ h⁻¹ mg⁻¹ drycell weight) were calculated by dividing volumetric rates by respectivevalues for cell mass.

Analyses. Organic acids and glucose were measured using a HewlettPackard HPLC (HP 1090 series II) equipped with a UV monitor (210 nm) andrefractive index detector. Products were separated using a Bio-RadHPX-87H column (10 μl injection) with 4 mM H₂SO₄ as the mobile phase(0.4 ml min⁻¹, 45° C.). Cell mass was estimated by measuring ODs_(550nm)(1.0 OD_(550nm) is equivalent to 0.33 g L⁻¹ dry cell weight) using aBausch & Lomb Spectronic 70 spectrophotometer and 10×75 mm culture tubesas cuvettes.

Results and Methods

Pyruvate as a co-product during acetate fermentations. Escherichia coliTC36 (pflB frdBC.ldhA.atpFH.adhE.sucA), as described above, wasengineered from W3110 (prototrophic) for the production of acetate (FIG.6A) by combining chromosomal deletions which minimize cell yield,fermentation products (reduced), oxygen consumption, and CO₂ evolution(Causey, T. B. et al. 2003). In this strain, glycolytic flux was 2-foldthat of the parent W3110 due to deletion of genes (atpFH) encoding twomembrane proteins that coupling the F₁ and F₀ components of theF₁F₀(H⁺)ATP synthase complex. This mutation eliminated ATP production byoxidative phosphorylation and also created an active, cytoplasmicF₁(H⁺)ATPase (FIGS. 6B and 6C). Glycolytic flux in TC36 exceeded thecapacity for acetate production under the conditions used for acetateproduction (5% air saturation at inoculation and during fermentation)resulting in the transient accumulation of approximately 16 mM pyruvatenear the end of exponential growth (FIG. 7).

By inoculating the fermentor at an initial dissolved oxygen level of100% air saturation (rather than 5% of saturation) and sparging with airuntil the oxygen level declined from 100% to 5% air saturation, thenadding oxygen to maintain 5% of air saturation, the peak level ofpyruvate of was increased to 81 mM (FIG. 7). Under these conditions,pyruvate yields were 25% of the maximum theoretical yield at the peakand 11% of the maximum theoretical yield at the end of fermentation whenglucose was fully metabolized (Table 5).

Effect of an acetate kinase (ackA) mutation on pyruvate production.Although there are many metabolic routes that can lead to acetate, theprimary catabolic routes for acetate production in E. Coli are theconversion of acetyl˜CoA to acetate by phosphotransacetylase (pta) andacetate kinase (ackA) and the direct oxidation of pyruvate to acetate bypyruvate oxidase (poxB) (FIG. 6A).

To block the acetate kinase route, strain TC38 was constructed from TC36by deleting the central region of the ackA gene. This additionaldeletion reduced the net production of ATP by 30% (FIG. 6A), cell yieldby 36% (FIG. 8A; Table 5), and the rate of growth by 45% (Table 6). Thismutation also reduced glycolytic flux by 45% (Table 6) and increased thetime required to complete fermentations from 18 h for TC36 to 24 h forTC44 (FIG. 8B). Acetate production was reduced by 85% (FIG. 8C; Table5), consistent with the acetate kinase pathway being the dominant routefor acetate production in TC36.

Although both volumetric and specific rates of glucose metabolism werelower for TC38 (Table 6), the pyruvate yield was 5.5-fold higher (Table5; FIG. 8D) and the specific rate of pyruvate production was 4-foldhigher (Table 6) than for TC36. Small amounts of 2-oxoglutarate,succinate, and fumarate were produced by both strains. From 10% to 15%of the carbon was not recovered as cell mass or acidic fermentationproducts and may have been lost as CO₂ due to metabolic cycling. Withstrain TC38, the pyruvate yield was 58% of the theoretical maximum.Acetate (28.9 mM) remained as the second most abundant product.

Effect of a pyruvate oxidase (poxB) mutation on pyruvate production.Pyruvate can be converted directly to acetate by the membrane-boundprotein pyruvate oxidase using the electron transport system to coupleoxygen as the terminal electron acceptor. The poxB gene is typicallyrepressed during exponential growth but is induced by stress or entryinto stationary phase (Chang, Y.-Y. and J. E. Cronan Jr. 1983; Chang,Y.-Y. et al. 1994).

Strain TC42 was constructed from TC36 by inserting a short DNA segmentcontaining stop codons into the central region of poxB. In contrast tothe ackA deletion (TC38), the poxB mutation (TC42) caused relativelysmall changes in metabolic products (Table 5) consistent with a minorrole for the PoxB pathway. Acetate levels for TC42 were 10% lower andpyruvate levels were higher than for TC36 (Table 5; FIGS. 8C and 8D).Although this represented a 2-fold improvement in pyruvate yield overTC36, the overall yield for pyruvate with TC42 was less than 30% of thetheoretical maximum (Table 5). These changes in metabolic products wouldhave little effect on ATP yields (FIG. 6A). Unlike the mutation in ackA,inactivation of poxB did not reduce the rate of growth or glucosemetabolism (FIG. 6A; Table 6).

Effect of combining mutations in pyruvate oxidase (poxB) and acetatekinase (acKA) on the production of pyruvate. To improve pyruvate yieldand reduce acetate production, strain TC44 (pflBfrdBC.ldhA.atpFH.adhE.sucA poxB::FRT.ackA) was constructed in which bothacetate kinase and pyruvate oxidase are inactive. Inactivation of poxBwas beneficial for growth and pyruvate production (FIG. 8A; Table 5 andTable 6) in comparison to TC38, an isogenic strain containing afunctional poxB. Adding the poxB mutation substantially restored bothvolumetric and specific rates of glucose metabolism to that observed forTC36 (Table 6) in which both acetate pathways are functional, whilefurther reducing acetate production. Acetate production by TC44 wasreduced by more than half in comparison to TC38 (acetate kinasedeletion) and pyruvate yield was increased by 17%. The specific rate ofpyruvate production by TC44 was 8-fold that of TC36 and twice that ofTC38 (Table 5). The time required to complete fermentation with TC44 was30% shorter than with TC 38 (FIG. 8B). Broth containing 3% glucose (167mM) was converted into 2.2% pyruvate (252 mM) after 18 h in mineralsalts medium (FIG. 8D). Although acetate levels were substantiallyreduced by the combining of poxB and ackA mutation (FIG. 8C), acetateand dicarboxylic acids remained as minor products.

The beneficial role of a poxB mutation for pyruvate production. Thepyruvate oxidase catalyzed oxidation of pyruvate to acetate (and CO₂)also contributes to the requirement for oxygen as an electron acceptor.Oxygen transfer rates are frequently limiting during aerobicfermentations at relatively high levels of saturation, and may be evenmore problematic under fermentation conditions (5% of air saturation).Eliminating the primary route for acetyl˜CoA dissimilation (ackA) inTC38 increased pyruvate production and may also increase the amount ofpyruvate that is metabolized by PoxB. Increasing oxygen saturation from5% to 50% during TC38 fermentations (Table 5 and Table 6) wasbeneficial. Cell yield, pyruvate yield, and the specific rate of glucosemetabolism were 8% to 41% higher for TC38 at 50% air saturation than at5% air saturation. These results were very similar to those observed forthe isogenic poxB mutant, TC44, during fermentation at 5% airsaturation. Increasing the oxygen saturation during TC38 fermentationsalso decreased the final concentrations of acetate to a level equivalentto TC44 at 5% air saturation and decreased the production ofdicarboxylic acids. As with TC44, low levels of acetate and dicarboxylicacids were also present at the end of fermentation with TC38 (50% airsaturation) (Table 5).

Improving pyruvate yields and titers in TC44 by altering fermentationconditions. With TC44 decreasing the ammonia level by half did notincrease product yields (Table 5). Doubling of the initial concentrationof glucose or providing a second addition of glucose (3% plus 3%)resulted in a small increase (11%) in yield accompanied by a 2-foldincrease in final pyruvate titer. The highest level of pyruvate, 749 mM,was produced with excess glucose. This may represent the limit forpyruvate tolerance. When pyruvate is added to minimal media at 600 mM,growth of wild type strains of E. coli is substantially inhibited.

In contrast to biocatalysts where vitamins and other complex nutrientsare required for effective production of pyruvate by fermentation, thenew biocatalyst of the subject invention, E. coli TC44, requires onlymineral salts and glucose. The lack of a requirement for vitaminsupplements, complex nutrients or complicated process controls for TC44provides a substantial savings in production costs. In addition, thelack of complex nutrients in the fermentation broth reduces costsassociated with product purification and waste disposal.

Pyruvate can be produced by a variety of microorganism including mutantsof yeasts and bacteria. However, E. coli TC44 provides a competitivealternative to the current pyruvate-producing biocatalysts due to highyields, high product titers, simple fermentation conditions, and theability to grow well in mineral salts medium with glucose as the solecarbon source (Table 7).

TABLE 5 Products formed from glucose catabolism by E. coli strainsdescribed herein. Carbon Product Concentrations (mM)^(a) Cell massBalance Pyruvate yield 2- Strain Condition Replicates (g · L⁻¹) (%) (%theoretical)^(b) Pyruvate Acetate Oxoglutarate Succinate Fumarate TC363% Glucose 3 3.64 ± 0.31 97.9 ± 4.8  0.31 ± 0.22  1.0 ± 0.7 223.8 ± 14.029.0 ± 23.7 4.6 ± 2.2 <0.1  5% DO^(c) TC36 3% Glucose 3 3.47 ± 0.23 89.0± 2.7 10.5 ± 7.9   38.1 ± 27.2^(j) 197.7 ± 21.1 16.6 ± 16.2 13.7 ± 13.21.4 ± 0.2 100->5% DO^(d) TC38 3% Glucose 3 2.21 ± 0.09 84.3 ± 5.2 57.5 ±2.6 194.5 ± 9.1  28.9 ± 16.7 10.5 ± 1.9  8.1 ± 9.1 0.8 ± 0.7 100->5%DO^(d) TC38 3% Glucose 2 2.40 84.7 68.8 241.9  7.0  7.9 nd^(k) nd^(k)100->50% DO^(e) TC42 3% Glucose 2 3.40 86.8 29.1  79.0 178.4  76.2 24.31.7 100->5% DO^(d) TC44 3% Glucose 3 2.36 ± 0.10 88.5 ± 0.6 69.3 ± 1.5252.5 ± 6.2 11.6 ± 1.2 3.6 ± 1.2 16.8 ± 0.7  1.1 ± 0.2 100->5% DO^(d)TC44 3% Glucose 2 2.02 73.6 38.8 125.2 50.3 30.0  7.7 2.9 ½ Nitrogen100->5% DO^(f) TC44 3 + 3% 2 2.63 86.7 72.3 479.8 39.8 31.7 10.9 0.7Glucose 100->5% DO^(g) TC44 6% Glucose 2 1.95 94.8 77.9 588.9 46.0 26.1nd^(k) 0.7 100->5% DO^(h) TC44 Excess Glucose 2 2.51 na^(l) na^(l) 749.0na^(l) 45.3 na^(l) 4.9 100->5% DO^(i) ^(a)Unless stated otherwise theconcentrations represent measurements at the time of complete glucoseconsumption. ^(b)Maximum theoretical yield is 2 moles pyruvate per moleglucose (0.978 g pyruvate g⁻¹ glucose). ^(c)3% glucose 10 L batchfermentation with the dissolved oxygen controlled at 5% of airsaturation by adjusting the ratio of O₂ and N₂ (Causey et al. 2003).^(d)3% glucose 5 L batch fermentation with the dissolved oxygen allowedto fall from 100% to 5% of air saturation. ^(e)3% glucose 5 L batchfermentation with the dissolved oxygen allowed to fall from 100% to 50%of air saturation. ^(f)3% glucose 5 L batch fermentation with thedissolved oxygen allowed to fall from 100% to 50% of air saturation. The(NH₄)₂PO₄ concentration was reduced to 1.25 g L⁻¹. ^(g)3% initialglucose 5 L batch fermentation with the addition of 3% glucose after 15h. The dissolved oxygen was allowed to fall from 100% to 50% of airsaturation. ^(h)6% glucose 5 L batch fermentation with the dissolvedoxygen allowed to fall from 100% to 50% of air saturation. ^(i)3%initial glucose 5 L batch fermentation with the automatic addition of590 ml of 4 M glucose over a period of 20 h. The dissolved oxygen wasallowed to fall from 100% to 50% of air saturation. ^(j)The maximumpyruvate concentration measured during glucose fermentations ranged from14.88 mM to 111.89 mM. Pyruvate excretion in TC36 is very sensitive todissolved oxygen, where elevated dissolved oxygen results in morepyruvate being excreted. The concentration of acetate at the time allglucose has been consumed depends on the amount of pyruvate produced.Pyruvate is rapidly converted to acetate after glucose is depleted. Thehigh standard deviations are a result of small differences in dissolvedoxygen concentrations between fermentors and co-metabolism of theexcreted pyruvate and glucose. ^(k)Not detected. ^(l)Not available

TABLE 6 Comparison of biocatalysts for pyruvate production. VolumetricPyruvate Relevant Carbon Nitrogen Fermentation [Pyruvate] ProductionYield Strain genotype/phenotype Source Source Time (h) (g · L⁻¹) (g ·L⁻¹ h⁻¹) (g · g⁻¹) Reference Candida lipolytica B₁ ⁻ Met⁻ glucose NH₄NO₃72 44 0.61 0.44 Footnote¹ AJ 14353 Debaryomyces B₁ ⁻ Bio⁻ glucosePeptone 96 42 0.44 0.42 Footnote¹ hansenii Y-256 Torulopsis B₁ ⁻ Bio⁻ B₆⁻ NA⁻ glucose Soybean 47 60 1.28 0.68 Footnote¹ glabrata acetate leakyhydrolysate ACII-3 (NH₄)₂SO₄ Torulopsis B₁ ⁻ Bio⁻ B₆ ⁻ NA⁻ glucose NH₄Cl56 69 1.23 0.62 Footnote¹ glabrata WSH-IP 303 Escherichia coli lipA2bgl⁺ atpA401 glucose Polypeptone 24 30 1.25 0.60 Footnote² TBLA-1Escherichia coli aceF fadR adhE ppc glucose Tryptone 36 35 0.97 0.65Footnote³ CGSC7916 acetate (NH₄)₂HPO₄ Escherichia coli pflB frdBC ldhAglucose (NH₄)₂HPO₄ 43 52 1.21 0.87 Described herein TC44 atpFH adhE sucAackA poxB ¹Li, Y., J. Chen, and S.-Y. Lun, and X. S. Rui, 2001“Efficient pyruvate production by a multi-vitamin auxotroph ofTorulopsis glabrata: key role and optimization of vitamin levels” Appl.Microbiol. Biotechnol. 55: 680-685. ²Yokota, A., Y. Terasawa, N.Takaoka, H. Shimizu, and F. Tomita, 1994 “Pyruvic acid production by anF₁-ATPase-defective mutant of Escherichia coli W1485lip2” Biosci.Biotech. Biochem. 58: 2164-2167. ³Tomar, A., M. A. Eiteman, and E.Altman, 2003 “The effect of acetate pathway mutations on the productionof pyruvate in Escherichia coli.” Appl. Microbiol. Biotechnol. 62:76-82.

TABLE 7 Comparison of metabolic rates. Glucose Consumption Rate PyruvateProduction Rate Specific^(b) Specific^(b) μ_(max) Volumetric^(a) (mmol ·L⁻¹ · Volumetric^(a) (mmol · L⁻¹ · Strain (h⁻¹) (mmol · L⁻¹ · h⁻¹) h⁻¹ ·g⁻¹ cdw) (mmol · L⁻¹ · h⁻¹) h⁻¹ · g⁻¹ cdw) TC36^(c) 0.49 ± 0.03 10.1 ±2.6 17.6 ± 1.5 nd^(f) nd^(f) (pflB frdBC ldhA atpFH adhE sucA) TC36^(d)0.51 ± 0.01 10.7 ± 0.9 29.7 ± 3.5 3.8 ± 3.0  5.3 ± 3.1 (pflB frdBC ldhAatpFH adhE sucA) TC38^(d) 0.28 ± 0.01  6.7 ± 0.6 16.3 ± 2.2 8.3 ± 0.721.1 ± 3.7 (pflB frdBC ldhA atpFH adhE sucA ackA) TC38^(e,g) 0.21  6 288 28 (pflB frdBC ldhA atpFH adhE sucA ackA) TC42^(d,g) 0.55 10 17 6 10(pflB frdBC ldhA atpFH adhE sucA poxB) TC44^(d) 0.34 ± 0.02  9.7 ± 0.727.2 ± 4.1 13.1 ± 0.3  40.4 ± 7.4 (pflB frdBC ldhA atpFH adhE sucA poxBackA) ^(a)Average volumetric rates of glucose utilization and pyruvateproduction. ^(b)Maximum specific rates of glucose utilization andpyruvate production per g dry cell weight (dcw). ^(c)3% glucose 10 Lbatch fermentation with the dissolved oxygen controlled at 5% of airsaturation by adjusting the ratio of O₂ and N₂. ^(d)3% glucose 5 L batchfermentation with the dissolved oxygen allowed to fall from 100% to 5%of air saturation. ^(c)Fermentation conducted with the dissolved oxygencontrolled at 50% of air saturation. ^(f)Not determined. ^(g)Average oftwo experiments.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A method for enhancing the microbial production of a desired productwherein said method comprises culturing a microbe having one or moregenetic modifications that reduce ATP, such that the microbe's sugarmetabolism is increased as is the rate of production of the desiredproduct.
 2. The method, according to claim 1, wherein saidmodification(s) decrease the amount of ATP produced during metabolism.3. The method, according to claim 1, wherein said modification(s)increase the rate of ATP consumption during metabolism.
 4. The method,according to claim 1, wherein said modification(s) decrease the amountof ATP produced during metabolism and increase the rate of ATPconsumption during metabolism.
 5. The method, according to claim 1,wherein said genetic modification(s) result in the elimination orsubstantial reduction of ATP production by oxidative phosphorylation. 6.The method, according to claim 4, wherein there is a retention ofcytoplasmic F_(i)-ATP synthase for consumption of ATP.
 7. The method,according to claim 1, wherein said microbe comprises geneticmodifications that inactivate oxidative phosphorylation, disrupt thecyclic function of the tricarboxylic acid cycle, and eliminate one ormore fermentation pathways.
 8. The method, according to claim 1, whereinthe microbe is a derivative of E. coli that comprises one or morechromosomal deletions selected from the group consisting of focA-pflB;frdBC; ldhA; atpFH; sucA and adhE.
 9. The method, according to claim 1,which comprises introducing into said microbe, one or more mutationsinto chromosomal genes thereby inactivating one or more pathwaysselected from the group consisting of lactate dehydrogenase, pyruvateformatelyase, fumarate reductase, ATP synthase, alcohol/aldehydedehydrogenase, and 2-ketoglutarate dehydrogenase.
 10. The method,according to claim 1, wherein the desired product is selected from thegroup consisting of acetic acid; 1,3-propanediol; 2,3-propanediol;pyruvate; dicarboxylic acids; adipic acid; amino acids; and alcohols.11. The method, according to claim 10, wherein said product is aceticacid.
 12. The method, according to claim 10, wherein said product ispyruvic acid.
 13. The method, according to claim 10, wherein said aminoacid is selected from the group consisting of aliphatic and aromaticamino acids.
 14. The method, according to claim 10, wherein said alcoholis selected from the group consisting of ethanol, butanol, isopropanoland propanol.
 15. The method, according to claim 1, wherein said microbeis an E. coli.
 16. The method, according to claim 1, wherein saidmicrobe is selected from the group consisting of TC36, TC24, TC44, andSZ47.
 17. The method, according to claim 16, wherein said microbe isTC36.
 18. The method, according to claim 16, wherein said microbe isTC44.
 19. The method, according to claim 1, wherein the desired productis produced via a natural pathway.
 20. The method, according to claim 1,wherein the desired product is produced via a recombinant pathway. 21.The method, according to claim 1, wherein said microbe is devoid ofplasmids and antibiotic resistance genes.
 22. The method, according toclaim 1, wherein said method comprises a two-step batch feeding strategywherein a second addition of glucose follows the end of an initialgrowth phase.
 23. The method, according to claim 22, wherein said methodfurther comprises a nitrogen limitation.
 24. A biocatalyst for acetateproduction wherein said biocatalyst is TC36.
 25. A biocatalyst forpyruvate production wherein said biocatalyst is TC44.